Quality control Flashcards
with a testing assay, what is difference between accuracy and precision?
Accuracy - same result as “gold standard” method
Precision - getting same result on each test
analogy is shooting 10 arrows at target. If all arrows grouped together, but miss target, this is precise, but not accurate
What are steps of each of these phases in quality assessment program?
Pre-analytic
Analytic
Post-analytic
Pre-analytic - test ordering processing order specimen collection/ transport specimen processing
Analytic -
examination of specimen
interpretation of results
Post-analytic -
formulation written report
institution of appropriate therapy
what is meaning of quality control in lab?
Laboratory quality can be defined as:
accuracy (same result pos or neg)
precision (same result specific value)
reliability (same result)
timeliness
of the reported test results.
Relies on set of procedures/ processes by the laboratory to ensure this
What are facets of quality control?
Test methods - specimen collection, transport, reagents, media, temperature logs
Verification/ validation - testing new methods
Procedures - SOPs
Personnel - training
What are the Westgard rules
13S
22s
41S
R4S
RX10
What is the Limit of Detection?
LoD is the lowest amount of a target or analyte that your assay can detect 95% of the time
e.g 25 copies
Could detect lower than 25 copies, however cannot do this reliably
What is the Limit of Quantification?
The Limit of Quantification (LOQ) is the lowest analyte concentration that can be quantitatively detected with a stated accuracy and precision
What is the difference between
Limit of Detection
Limit of Quantification
LoD is lowest amount of a target or analyte that your assay can detect 95% of the time - can detect, but cannot quantify
LoQ is the lowest concentration at which the analyte can not only be reliably detected, but at which some predefined goals for bias and imprecision are met
What is the threshold for PCR?
The threshold of the real-time PCR reaction is the level
of signal that reflects a statistically significant increase
over the calculated baseline signal.
It is set to distinguish relevant amplification signal from the
background. Usually, real-time PCR instrument software
automatically sets the threshold at 10 times the standard
deviation of the fluorescence value of the baseline.
However, the positioning of the threshold can be set at any
point in the exponential phase of PCR.
What is the definition of threshold cycle
The threshold cycle (Ct) is the cycle number at which the
fluorescent signal of the reaction crosses the threshold.
The Ct is used to calculate the initial DNA copy number,
because the Ct value is inversely related to the starting
amount of target.
For example, in comparing real-time PCR results from samples containing different amounts of target, a sample with twice the starting amount will yield a Ct one cycle earlier than a sample that contained half as many copies of the target prior to amplification
What are viral load calibrators?
Have known concentrations of a virus e.g
100 copies
1000 copies
10000 copies
Compare fluorescence of your sample to calibrators, to calculate the number of copies in your sample
Need minimum 5x values
What is PCR efficiency?
Each cycle should have doubling of DNA - 100% efficiency
If inhibitors present, or not enough reagents - efficiency can drop
What is the R2 (R-squared) value?
R2 is the correlation coeffient
R2 is a statistical term that indicates how good one value is at predicting another - reflects how linear the standard curve is
When R2 is 1, the value of Y (Ct) can be used to accurately predict the value of X value
An R2 value >0.99 provides good confidence in correlating two values.
R2 >0.99 means there is a nice straight line of correlation. That means if we increase Ct value from 1 to 2, then we know that the viral load value will double
What is measurement uncertainty (MU)?
Measurement Uncertainty (MU) relates to the margin of doubt that exists for the result of any measurement, as well as how significant the doubt is. For example, a piece of string may measure 20 cm plus or minus 1 cm, at the 95% confidence level. As a result, this could be written: 20 cm ±1 cm, with a confidence of 95%. Therefore, we are 95% sure that the piece of string is between 19 cm and 21 cm long.
Understanding this measurement
uncertainty assists in interpreting this
result, and understanding if there is a real
difference between this and a future result.
Medical pathology laboratories are required as part of their accreditation to determine or estimate the
MU for all quantitative results.
For a test to be useful, measurement of uncertainty must fall greatly below the normal variation. e.g if normal CRP is 10, then MU must be 1-5 so it does not overlap with “normal”
What steps are taken to estimate measurement of uncertainty?
Estimating MU in a laboratory environment is a four step process involving the following:
- Define what will be measured.
- Identify all sources of uncertainty.
- Quantify the uncertainties of each of these sources.
- Calculate the combined uncertainty.
(It is usual practice to calculate the 95% confidence interval for pathology measurements, calculated as 1.96 standard deviations or 1.96 coefficients of variation about the measured value.)
Use IHC quality control samples to check that that analysis is acceptable against predetermined values
For example a viral load is 804, and repeat is 870. These two values fall within same measurement of uncertainty, so should be considered the same value
