Within- and Between-Operator Variability in Real-Time PCR-Based Human DNA Quantification and the Effect of Applying Calibration Curves Generated in Different Runs: A Preliminary Single-System Study

Background/Objectives: Quantification of human DNA before short tandem repeat (STR) typing is an essential step in forensic DNA analysis, and the DNA quantity recovered from trace material can itself be weighed in court, as illustrated by a recently concluded Japanese criminal case in which the amount of DNA on a complainant’s skin was a central point of contention. For one specific instrument–assay combination, we characterized within- and between-operator variability of real-time PCR quantification and quantified how much an estimate changes when the calibration curve used to convert it originates from a different run. Methods: Three examiners quantified three oral-swab DNA extracts from three donors (low, medium and high concentration) on a SmartCycler II with a human genomic DNA quantification kit (Ver. 2), each reaction in duplicate, over four consecutive days and, separately, in five consecutive runs within one day (27 runs, 162 unknown measurements). Each unknown quantification cycle (Cq) was then re-converted, with its Cq held constant, using the calibration curve of every other run in the same experiment block (2052 paired comparisons), stratified by whether the operator and the day changed. Results: All 27 curves had R-squared ≥ 0.995, yet slopes ranged from −3.13 to −3.90 (amplification efficiency 80.4–108.8%), so a single Cq of 16.80 mapped to 0.51–1.22 ng/μL, a 2.4-fold range, according only to which run supplied the curve. Coefficients of variation per sample and operator were 7.6–19.5% between days and 7.3–18.7% within a day; repeatability CV was 4.9–13.7% and intermediate-precision CV 10.9–17.6%. Applying a non-contemporaneous curve changed the estimate by a median of 14.7% (interquartile range 7.0–27.6) when the curve came from another run by the same operator on the same day, 13.6% (6.3–20.7) when it came from a run by the same operator on a different day, and 17.7–18.6% (8.5–30.1) for cross-operator pairings, with single changes up to 102%. Changes occurred in both directions within every stratum; mean signed changes of 1.9–3.3% are reported descriptively. Conclusions: In this system, applying a calibration curve generated in a different run changed the reported concentration by a median of approximately 14–19%. Within the same operator, separation of the runs by days rather than minutes was not associated with a larger discrepancy. Because the three examiners measured sequentially and never on the same day, operator identity was fully confounded with calendar period, so operator-specific effects could not be estimated independently and the larger cross-operator values are reported descriptively only. R-squared alone does not reveal this cross-run variability. These findings support the generation and preservation of run-specific calibration data whenever quantitative DNA results may carry evidential weight.