Limit of Detection: The Smallest Signal a Molecular Assay Can Call

What the Limit of Detection Is

The limit of detection is the lowest amount of a target, usually expressed as a variant allele fraction, that the assay can detect with a stated probability, commonly 95 percent, while keeping false positives acceptably low.

It is established during validation by testing dilution series of known-positive material and is a property of the assay for a given variant type and input amount.

What Sets It

Key factors are sequencing depth (more reads distinguish a true low-level signal from error), the background error rate of the chemistry and bioinformatics, the amount and quality of input DNA, and the variant type.

Point mutations generally have the lowest limits of detection; insertions, deletions, copy-number changes and fusions are harder and have higher (worse) limits.

Tissue vs Blood

Standard tumour tissue panels often have limits of detection around 2 to 5 percent variant allele fraction. Specialised circulating tumour DNA assays push far lower, to 0.1 percent or below, using error correction and by tracking many variants together.

Ultra-sensitive residual-disease assays achieve their sensitivity partly by being personalised to a known set of tumour mutations rather than searching broadly.

Why It Governs Negative Results

If a mutation is present at 1 percent and the assay's limit of detection is 5 percent, the assay will report negative even though the mutation is there. The negative is accurate for the assay but not for the tumour.

This is why a clinically expected mutation that is not found should be reconsidered in light of sample purity and the assay's limit of detection before concluding true absence.

Interpretation Notes

A report should state the assay's limit of detection, ideally by variant type. Treat a negative result as ruling out the variant only above that threshold.

For low-tumour-content samples or blood-based tests, the effective sensitivity for a specific case can be worse than the headline limit of detection.

Key Takeaways

• ·Limit of detection is the lowest variant fraction an assay reliably calls, set during validation.
• ·Depth, error rate, input quality and variant type all influence it.
• ·Tissue panels sit around 2 to 5 percent; specialised ctDNA assays reach 0.1 percent or lower.
• ·A negative result only excludes the variant above the assay's limit of detection.