Limit of Blank, Limit of Detection and Limit of Quantitation
*David A Armbruster,1
Terry Pry 2
1Global Scientific Affairs, Abbott Diagnostics, Abbott Park, IL 60064, USA, 2
Formerly ANZ Scientific Affairs, Abbott Diagnostics, Mt Wellington, Auckland, New Zealand.*For correspondence: Dr Dave Armbruster e-mail: David.Armbruster@abbott
Summary
•  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.•  LoB is the highest apparent  analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested.  LoB = mean blank + 1.645(SD blank )•  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. LoD = LoB + 1.645(SD low concentration sample )
•  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. Introduction Sensitivity, Analytical Sensitivity, Functional Sensitivity, Lower Limit of Detection, LoB, LoD, and LoQ are terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure. There has often been a lack of agreement within the clinical laboratory field as to the terminology best suited to describe this parameter. Likewise, there have been various methods for estimating it. Clinical laboratorians have perhaps been lax in dealing with this analytical issue because, in many cases, the ability of a laboratory test to detect a very small amount of measurand is not clinically significant. For example, the medical decision levels for glucose and cholesterol are so far above the lower analytical limits of these tests that it is highly unlikely that clinical action will depend on measurements of these analytes at such low concentration. Nevertheless, it is important to fully characterise the analytical performance of every clinical laboratory test in order to understand its capability and limitations, and to ensure that it is “fit for purpose.” Moreover, defining the limits of an assay at low concentration is directly related to its dynamic range, or analytical measurement range.To provide a standard method for determining LoB, LoD and LoQ, Clinical and Laboratory Standards Institute (CLSI) has published the guideline EP17, Protocols for Determination of Limits of Detection and Limits of Quantitation.1  The
Figure taken from their document illustrates the distinction of LoB, LoD and LoQ values. Typically, LoQ will be found at a higher concentration than LoD, but how much higher depends on the specifications for bias and imprecision used to define it. ‘Analytical sensitivity’ defined as the slope of the calibration curve is sometimes used as a synonym for LoD. However, because LoD may well reside at some concentration below the linear range of an assay, where the calibration curve is no longer valid, this usage should be avoided.All of these parameters are related but have distinct definitions and should not be confused. The intent is to define the smallest concentration of analyte that can be detected  with no guarantee about the bias or imprecision of the result by an assay, the concentration at which quantitation as defined by bias and precision goals is feasible, and finally the concentration at which the analyte can be quantitated with a linear response.Limit of Blank EP17 defines LoB as the highest apparent  analyte concen-tration expected to be found when replicates of a sample containing no analyte are tested.1 Note that although the samples tested to define LoB are devoid of analyte, a blank
LoB/LoD/LoQ
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.1
Limit of Quantitation
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).5It 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 pur
pose”) 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.
The Table provides a brief summary of the features of the LoB, LoD, and LoQ.
Conclusions
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.
characterise
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
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Figure. Relationship between LoB, LoD and LoQ. The solid line defines the LoB and represents the dis
tribution of results for a blank specimen. As modern analysers seldom report results of less than zero, the frequency of “zero” results is artificially increased as illustrated. The LoB shown excludes a small proportion of blank results (“α”). The dashed line defines the LoD and represents the scatter (imprecision) of results for a specimen of low concentration. The LoD is then set so that only a small proportion (“β”) of these results will fall below LoB. The dotted line defines the LoQ and represents the distribution of results for a specimen of low concentration meeting the target for total error (imprecision and bias). It may be that this LoQ total target error is met by a specimen at the LoD concentration in which case LoQ = LoD. Otherwise, LoQ will have to be set after testing a specimen of higher concentration. Reproduced with permission, from NCCLS publication EP17-A, Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline.

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