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Limit of Detection or Limit of Quantitation, how to differentiate?

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Limit of Detection or Limit of Quantitation, how to differentiate?

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Summy


Limit of Blank (LoB), Limit of Detection (LoD), and Limit of Quantitation (LoQ) are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure.




LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte.


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. The LoQ may be equivalent to the LoD or it could be at a much higher concentration.


Limit of Detection


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Although reagent package inserts may state that an assay has a dynamic range that extends from zero concentration to some upper limit, typically an assay is simply not capable of accurately measuring analyte concentrations down to zero. Sufficient analyte concentration must be present to produce an analytical signal that can reliably be distinguished from “analytical noise,” the signal produced in the absence of analyte.


LoD is the lowest analyte concentration likely to be reliably distinguished from the LoB and at which detection is feasible. It is therefore greater than LoB (Figure). A traditional and typical approach to estimate LoD consists of measuring replicates, usually n=20, of a zero calibrator or blank sample, determining the mean value and SD, and calculating LoD as the mean +2 SD. Variations of this approach use the mean plus 3, 4, or even 10 SDs to provide a more conservative LoD. The assumption is that if analyte is present, it will produce a signal greater than the analytical noise in the absence of analyte. This is a simple and quick method. The weakness is that there is no objective evidence to prove that a low concentration of analyte will indeed produce a signal distinguishable from a blank (zero concentration) sample. As Needleman and Romberg noted, “It defines only the ability to measure nothing”.2


An alternative approach utilises analysis of samples containing small but known concentrations of the substance of interest (be it a drug, hormone or other analyte).3,4 The advantage of this empirical approach is that objective data is used to compare the analytical response of blank and low concentration samples to determine conclusively what concentration of analyte is necessary to distinguish its presence from its absence. Various analytical specifications (e.g. a minimum signal-to-noise ratio for a chromatographic method or a minimum absorbance requirement for a spectrophotometric procedure) can be applied to ensure that the LoD is meaningful and clearly distinguishable from a negative or blank sample.

As defined in EP17, LoD is determined by utilising both the measured LoB and test replicates of a sample known to contain a low concentration of analyte.1 The mean and SD of the low concentration sample is then calculated according to:


LoD = LoB + 1.645(SDlow concentration sample)


Again assuming a Gaussian distribution of the low concentration samples, 95% of values will exceed the previously defined LoB, and only 5% of low concentration samples will produce values below the LoB and erroneously appear to contain no analyte. Manufacturers are expected to establish the LoB and LoD using two or more instruments and reagent lots to capture the expected performance of the typical population of analysers and reagents. A recommended practical number of LoB and LoD samples to be used by a manufacturer to establish these parameters is 60, while a laboratory verifying a manufacturer’s LoD (and possibly the LoB) is 20.


Once a provisional LoD is established, it can be confirmed by examining the observed values for samples containing the LoD concentration. Some LoD sample values are expected to be less than the estimated LoD (Figure), but when using 1.645 SD, no more than 5% of the values should be less than the LoB. If the observed LoD sample values meet this criterion, the LoD is considered established or verified. If more than 5% (roughly 1 out of 20 observations) of the LoD sample values fall below LoB, the LoD is too low and must be re-estimated (i.e. by testing a sample of higher concentration that will generate a higher mean and SD and thus a higher LoD).


This is an abbreviated and simplified description of the EP17 protocol. The guideline contains considerably more statistical detail and guidance, including the use of non-parametric (non-Gaussian) techniques if necessary. Readers are encouraged to consult EP17 for a complete explanation of this method for establishing and verifying LoD.


Limit of Quantitation

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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. “Functional sensitivity” is defined as the concentration that results in a CV=20% (or some other predetermined % CV), and is thus a measure of an assay’s precision at low analyte levels (without addressing bias).5 It was originally developed as a clinical diagnostic tool to characterise thyroid stimulating hormone (TSH) assay performance in distinguishing euthyroid from hyperthyroid patients at low TSH concentrations. It can be expected that the LoD lies somewhere below an assay’s functional sensitivity.


The LoQ may be equivalent to the LoD or it could be at a much higher concentration (Figure); it cannot be lower than the LoD. A LoD provides an estimate of bias and imprecision at very low analyte concentration. If the observed bias and imprecision at the LoD meet the requirements for total error for the analyte (i.e. the assay is “fit for purpose”) then: LoQ=LoD. If the analytical goals are not met at the LoD, a slightly higher analyte concentration must be tested to determine the LoQ.


Conclusions

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It is important to fully characterise the analytical performance of clinical laboratory tests in order to understand their capability and limitations, and to ensure that they are “fit for purpose.” The terms LoB, LoD, and LoQ describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure.


To establish these parameters a manufacturer would test a large number of sample replicates to increase the robustness and the statistical confidence of the estimate (Table 1). In addition, a manufacturer establishing the LoB, LoD, or LoQ should perform studies using more than one analyser and one lot of reagents to encompass the variability that users can expect to encounter in the field. Clinical laboratories can validate these parameters using a smaller number of samples and likely will use only one analyser and one lot of reagents.


LoB and LoD are important for tests used to discriminate between the presence or absence of an analyte (e.g. drugs, troponin, human chorionic gonadotrophin) and LoQ, to reliably measure low levels of hormones (e.g. TSH) for clinical diagnosis and management and should be incorporated as part of any method evaluation.


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