Principles of analysis Flashcards
What are the 3 phases of testing?
pre analytical
analytical
post analytical
Match the following
A. Pre analytical
B. Analytical
C. Post analytical
- reference ranges, diagnostic sensitivity and specificity
- Methodology, calibraiton, assessment of assay performance, quality control
- sample variables and patient variables
C1, B2, A3
What is a definitive method?
Method of exceptional scientific summary suitable for certification of reference material
What is a reference method?
Method demonstrating small inaccuracies against definitive method.
Method to test the performance of the definitive method
What is a routine method?
Method deemed sufficiently accurate for routine use against reference method and standard reference materials.
Match the following methodology terms?
A. Reference method
B. Definitive method
C. Routine method
- Method of exceptional scientific summary suitable for certification of reference material
- Method deemed sufficiently accurate for routine use against reference method and standard reference materials.
- Method demonstrating small inaccuracies against definitive method.
C2, B1, A3
Use reference, definitive, routine method with an example of cholesterol
Definitive method: isotope dilution/gas chromatography/mass spectrometry
Reference method: abell- Kendall Method
- hydrolysis of cholesterol esters with alcoholic KOH
- extraction of total cholesterol with hexane for 15 mins -> dried in vacuum
- treated with acetic acid/acetic anhydride/sulphuric acid 30min then abs at 620nm
Routine method: enzymatic e.g. Beckman
What is a primary standard?
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 standard reference material (SRM)?
Reference material issued by an institute whose values are certified by a reference method which establishes traceability
aka. Material produced which has been established as useful by reference method and established traceability
What is a secondary standard?
A commercially produced standard for routine use calibrated against a primary standard or reference material
What can calibrators give us clinicians?
Allows us to produce a standard curve when we are measuring something being emitted. This means we can confidently quantify the amount of a certain substance.
Allows the relationship of analyte conc and signal to be examined
Requirements of calibrators.
Prepared from pure substance
Stable and homogenous material
Matrix similar to assay matrix e.g. serum
If possible should be obtained commercially to min error
Match the following, calibrator values:
A. Stated value
B. Assigned value
C. Certified value
D. Standard reference method value
- derived using a reference method
- No certification
- Given arbitrarily or derived using non-reference method
- certification of value by particular institute or body
D1, B3, C4, A2
Define traceability
An unbroken chain of comparisons of measurements leading to a reference value
Importance of traceability
Ensures reasonable agreement between routine and reference method
What makes a good method?
Analytical accuracy
Analytical precision
Match
A. Analytical accuracy
B. Analytical precision
- Measure of agreement between a measured quantity and true value.
- Measure of agreement between replicates.
A1, B2
How is precisiona and accuracy implicated in screening?
Important to avoid FN and FP
Accuracy is a combination of what and involves what?
combination of trueness and precision
involves systemic and random error
What is a random error?
Random deviation from expected value due to inaccurate pipetting, sample contamination
Systemic error
Shift in expected values indicating bias
What does trueness measure?
systemic error or bias
How can degree of precision can be measured?
by quantifying the overall effect of all random errors
Mehtods of testing trueness?
Recovery experiment
Comparison to fresh EQA material
Correlation with a current/accepted method using patient samples
Value for precision?
Coefficient of variation = SD/M x100%
- Allows comparison between results from different analyte in a way stdev cant
- Ideal CV <5%
What is an interference?
Specific component that can be identified as causing an effect
Difference between serum and plasma sample collection.
Serum is a clotted blood sample and is therefore devoid of clotting factor (no anticogulant). Plasma anticoagulated before centrifuge
Why is blood an appropriate sample for testing?
Convient, taken randomly
Detect; leakage from damage tissue, hormones, waste, metabolites not properly controlled, nutrient levels
Urine as a sample?
Natural waste product of body
Measures: electrolyrs, nitrogenous compounds, phosphates, drug metabolims
Levels of hydration; ratio e.g. to creatinine
Dehydrated indicated by low creatinine
Cerebral spinal fluid as sample?
Difficult to obtain, but very good for specific conditions
- secretory product of ventricels, choroid plexus
Levels of typical components are similar to those found in blood or plasma
What are some blood preservatives and what do they do?
Required to prevent metabolism of certain analytes in vivo- can interfere with assays
- potassium EDTA (anticoagulant)
- lithium heparin (blocks clotting)
- sodium citrate
- fluoride oxalate
What is haemolysis?
Disruption of the membrane of red blood cells resulting in release of cellular contents - most common intereference
What percentage of people fall within +/-1std off the mean?
68%
What percentage of people fall within +/-2std off the mean?
95% this is within the reference range
Match the following calculations of reference ranges.
A. Parametric method
B. Non-parametric-method
C. Target driven
- Makes no assumption about type of distribution
- Reference range cant be derived from healthy pop
- Assumes gaussian distribution
A3, B1, C2
What is excluded in a reference range production?
People with Disease, Risk factors, Intake of pharmacological agents, physiological states
What is the total CV calculation?
CV(T)=sqrt CV^2(ana)+CV^2(biological)
Define clinical sensitivity
Ability of a test to correctly identify those who have the disease - highly sensitive = few false negatives
Define clinical specificity
Ability of a test to correctly identify those who do not have a disease
- highly specific = few false positives
What is predictive value?
measure of the ability of test to correctly assign individual to either disease or non-diseased group
What is diagnostic efficiency?
proportion of true results
Match the following to what describes the the best.
A. Clinical specificity
B. Clinical sensitivity
C. Diagnostic efficiency
D. Predictive value
- Proportion of true results
- Ability of test to correctly ID those without disease
- Ability of test to correctly ID people with disease
- Ability of test to correcltly assign either disease or non
A2
B3
C1
D4
With regards to the 2x2 table how is sensitivity, specificity, positive/negative predictive value and diagnostic efficiency calculated
Sensitivity = TP/(TP+FN) Specifity = TN/(TN+FP)
PPV= TP/(TP+FP)
PPN=TN/(TN+FN)
How is analytical quality monitored as a whole?
In real time using an internal quality control (IQC)
Retrospectively through the use of external quality assurance (EQA)
Explain IQC.
IQC monitors the performance of an assay. Will the same assay produce the same result multiple times? The expected value should be known and if its not obtained the assay should be reconsidered before releasing results to a patient
Explain EQC
Allows for retrospective analysis of performance of an assay between other users. Are the results the same in other labs using same analyser or assay?
What are some basic parameters involved in choosing IQC material?
Matrix
- close matrix match. Human were possible
Third party
- min one QC should not be made by assay manufacturer. To minimise bias.
Levels available
- min 2- one normal(physiological) and high (pathological)
Range of analytes
- QC multiple assays
Cost - cost effectiveness without compromising quality
Stability - repeatability
How often is IQC needed?
Depend on frequency of analysis
- Batch analysis: required at beginning and end
- continuous flow requires throughout
Accuracy of results between acceptable IQC results
Depends on analyte
