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Clinical Biochemistry: Analytical Methods > Lecture 1: Principles of Analysis > Flashcards

Lecture 1: Principles of Analysis Flashcards

1
Q

What is a Definitive Method?

A

A method of exceptional scientific accuracy suitable for certification of reference material

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2
Q

What is a reference method?

A

Method demonstrating small inaccuracies against definitive method

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3
Q

What is a routine method?

A

Method deemed sufficiently accurate for routine use against reference method and standard reference materials

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4
Q

What is a Primary Standard?

A

A substance of known chemical composition and high purity that can be accurately quantified and used for assigning values to materials and calibrating apparatus

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5
Q

What is a Standard Reference Material?

A

Reference material issued by an institute whose values are certified by a reference method which establishes tracebility

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6
Q

What is a Secondary Standard?

A

Commercially produced standard for routine use calibrated agaist a primary standard or reference material

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7
Q

What is an Internal Standard?

A

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
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8
Q

What are calibrator requirements?

A
  • 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
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9
Q

What are definitions of calibrator values?

A
  • 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
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10
Q

What is traceabillity?

A

An unbroken chan of comparisons of measurements leading to a reference value

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11
Q

What is Analytical Accuracy and Analytical Precision?

A
  • 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

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12
Q

What are clinical uses of results?

A
  • 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
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13
Q

Why is precision and accuracy important for screening?

A

Screening is important to avoid false positives and false negatives

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14
Q

What is verification and validation?

A
  • 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
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15
Q

What is accuracy?

A

Improving accuracy is a combination of improving trueness and improving precision

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16
Q

What is Trueness?

A

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
17
Q

How is trueness measured?

A
  • 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)
18
Q

How is Precision measured?

A

Degree of precision can be measured by quantifying the overall effect of all random errors.

2 Forms:

  • Repeatability
  • Intermediate precision
19
Q

What is repeatability?

A

Minimum 20 results obtained from repeat analysis of IQC and patient samples on the same run

20
Q

What is intermediate precision?

A

Minimum 20 results obtained from repeat analysis of IQC and patient samples from runs on different days

21
Q

What is the equation for measurement of precision?

A

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
22
Q

How is Accuracy measured?

A

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

23
Q

What is inteference?

A

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
24
Q

What is analytical specificity and analytical sensitivity?

A
  • Analytical Specificity: Measure of a method to determine only the analyte of interest
  • Analytical Sensitivity: Ability of a method to detect small concentrations
25
Q

What is Limit of Blank?

A

Highest measurement result that is likely to be observed (with a stated probability) for a blank sample

26
Q

What is the Limit of Detection?

A

Lowest amount of analyte in a sample that can be detected with (stated) probability, although perhaps not quantified as an exact value

27
Q

What is the Limit of Quantification?

A
  • 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
28
Q

What is Functional Sensitivity?

A

Functional sensitivity is defined as the lowest concentration that can be measured with acceptable intermediate precision

29
Q

What is the measuring interval?

A
  • 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
30
Q

What is Linearity?

A
  • 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
31
Q

What is a reference range?

A
  • 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
32
Q

How are reference ranges calculated?

A
  • 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
33
Q

What are some exclusion criteria from reference ranges?

A
  • 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
34
Q

What is Biological variation and the equation for it?

A
  • 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

35
Q

What is Clinical Sensitivity and Clinical Specificity?

A
  • 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
36
Q

What is Predictive Value?

A

Predictive value (+/-) = Measure of the ability of test to correctly assign individual to either disease or non-diseased group

37
Q

What is diagnositic efficiency?

A

Diagnostic efficiency = proportion of true results

38
Q
A

Clinical Biochemistry: Analytical Methods (2 decks)

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