Lecture 1: Principles of Analysis

Question 1

What is a Definitive Method?

Answer 1

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

Question 2

What is a reference method?

Answer 2

Method demonstrating small inaccuracies against definitive method

Question 3

What is a routine method?

Answer 3

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

Question 4

What is a Primary Standard?

Answer 4

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

Question 5

What is a Standard Reference Material?

Answer 5

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

Question 6

What is a Secondary Standard?

Answer 6

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

Question 7

What is an Internal Standard?

Answer 7

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

Question 8

What are calibrator requirements?

Answer 8

• 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

Question 9

What are definitions of calibrator values?

Answer 9

• 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

Question 10

What is traceabillity?

Answer 10

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

Question 11

What is Analytical Accuracy and Analytical Precision?

Answer 11

• 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

Question 12

What are clinical uses of results?

Answer 12

• 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

Question 13

Why is precision and accuracy important for screening?

Answer 13

Screening is important to avoid false positives and false negatives

Question 14

What is verification and validation?

Answer 14

• 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

Question 15

What is accuracy?

Answer 15

Improving accuracy is a combination of improving trueness and improving precision

Question 16

What is Trueness?

Answer 16

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

Question 17

How is trueness measured?

Answer 17

• 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)

Question 18

How is Precision measured?

Answer 18

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

2 Forms:

• Repeatability
• Intermediate precision

Question 19

What is repeatability?

Answer 19

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

Question 20

What is intermediate precision?

Answer 20

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

Question 21

What is the equation for measurement of precision?

Answer 21

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

Question 22

How is Accuracy measured?

Answer 22

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

Question 23

What is inteference?

Answer 23

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

Question 24

What is analytical specificity and analytical sensitivity?

Answer 24

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

Question 25

What is Limit of Blank?

Answer 25

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

Question 26

What is the Limit of Detection?

Answer 26

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

Question 27

What is the Limit of Quantification?

Answer 27

* 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

Question 28

What is Functional Sensitivity?

Answer 28

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

Question 29

What is the measuring interval?

Answer 29

* 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

Question 30

What is Linearity?

Answer 30

* 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

Question 31

What is a reference range?

Answer 31

* 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

Question 32

How are reference ranges calculated?

Answer 32

* **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

Question 33

What are some exclusion criteria from reference ranges?

Answer 33

* 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

Question 34

What is Biological variation and the equation for it?

Answer 34

* 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**

Question 35

What is Clinical Sensitivity and Clinical Specificity?

Answer 35

* **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

Question 36

What is Predictive Value?

Answer 36

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

Question 37

What is diagnositic efficiency?

Answer 37

Diagnostic efficiency = proportion of true results