CLSI EP17-A protocol: A useful tool for better understanding the low end performance of total prostate-specific antigen assays☆,☆☆
Introduction
Low end precision performances are very important for laboratory methods, especially when their results have clinical significance at these levels. Traditionally, developers claim in product inserts the “analytical sensitivity”, defined as the minimum detectable amount of analyte measured with a zero standard replicate-based protocol, and “functional sensitivity”, defined as the analyte concentration corresponding to 20% coefficient of variation (CV) measured with an imprecision profile protocol. The initial imprecision profile protocol was recommended for functional sensitivity (20% CV) determination and became a model broadly adopted by developers of analytes like thyrotropin and prostate-specific antigen (PSA). The concentration corresponding to 10% CV is a new goal for Troponin functional sensitivity, and is used in conjunction with a 99th percentile URL cut-off [1], [2].
In 2004, the National Committee for Clinical Laboratory Standards (NCCLS), now the Clinical Laboratory and Standards Institute (CLSI), published the EP17-A approved guideline: “Protocols for Determination of Limits of Detection and Limits of Quantitation” [3]. This protocol recommends new definitions for low end performances: Limit of Blank, Limit of Detection and Limit of Quantitation, because “sensitivity” or “analytical sensitivity” are used interchangeably with “limit of detection”, “lower LoD” or “detection limit” and misunderstandings can also occur between “functional sensitivity”, “clinical sensitivity”, “cut-off” and “critical value”.
Limit of Blank (LoB) is thereby defined as:“ the highest value we expect to see in a series of results on samples that contain no analyte”; limit of Detection (LoD) as “the actual concentration at which an observed test result is very likely to exceed the LoB and may therefore be declared as detected”, whereas limit of Quantitation (LoQ) is defined as “the actual concentration at which the analyte is reliably detected and at which the uncertainty of the observed test result is less than or equal to the goal set by the laboratory”.
Access®Hybritech® Total PSA is a chemiluminescent assay for total PSA detection. The same reagent can be used on different Beckman Coulter platforms and can be calibrated against two standards, Hybritech and World Health Organization (WHO). Two recent studies evaluated the clinical impact of a new WHO standardised prostate-specific antigen assay on biopsy rates and cancer detection [4] and in prostate cancer screening [5] .
The assay product insert states different cut-offs for the Hybritech and WHO calibrations at 4.0 and 3.1 μg/L respectively, but identical analytical (0.008 μg/L) and functional sensitivity (0.019 μg/L).
The aim of this study was to use the EP17-A protocol to determine the LoB, LoD and LoQ for the Access Hybritech Total PSA assay calibrated to both the Hybritech and WHO standards, and to use samples collected for the study to verify the correlation between both calibrations, particularly at low end concentrations.
Section snippets
Materials and methods
All pools were prepared using anonymous routine residual material from patient samples collected in Vacuette® 5 mL Serum Sep Clot Activator Becton–Dickinson Vacutainers. After an appropriate clotting time, tubes were centrifuged for 6 min at 3500 rpm at 18 °C, pooled in 500-μl aliquots and stored at 4 °C (for the LoB and LoD) or − 80 °C (for the imprecision profile and LoQ).
LoB was assayed with six samples (S1–S6, using 10 duplicates over 10 days): S1 and S2 were zero calibrators without PSA from two
Results
LoB results were 0.0046 μg/L with Hybritech calibration and 0.005 μg/L with WHO calibration. The calculated LoD was 0.014 μg/L with Hybritech calibration and 0.015 μg/L with WHO calibration.
Using 10% CV as the analytical precision goal, the LoQ was 0.0414 μg/L with Hybritech calibration and 0.034 μg/L with WHO calibration. Using 20% CV, the LoQ was 0.0152 μg/L with Hybritech calibration and 0.014 μg/L with WHO calibration. Hyperbolic and linear imprecision profiles are shown in Fig. 1.
Regression
Discussion
Laboratory methods have many performance characteristics that must be understood and verified before use. The product inserts provided by some manufacturers lack detailed information on these characteristics.
Limits of detection and quantitation are critical and important for many parameters, especially when the results have clinical significance at low levels, as in the case of troponin and tumor markers like PSA. Even though the EP 17-A protocol is fairly complex and is intended primarily for
Acknowledgments
We thank Beckman Coulter Italy for supplying PSA tests and M.C. Anelli for scientific support.
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The work was done in the Laboratory of Clinical Pathology “S. Croce Hospital” Fano (PU) Italy.
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We thank Beckman Coulter Italy for supplying the PSA reagents and M.C. Anelli for scientific support.