If you observe…

Check this metric first:

Likely cause:

Low Z’ but high Robust Z’

Classical vs. Robust Z’ comparison

Outlier wells in control population

High Neutral RSDE

Fluorescence CV of reference wells

Inconsistent dye loading or sample variability

Low S/N on titration curve

Response Amplitude (Δ Ratio)

Insufficient assay window; revisit target/dye concentration

High Δ Fluorescence (titration or single point)

650 nm fluorescence across all wells

Compound autofluorescence, quenching, or aggregation

Many poor reads

Well scan quality scores

Capillary blockage, precipitation, or instrument issue

|SSMD| < 6 but Z’ ≥ 0.5

Control population variances

Unequal variances between positive control and reference

Marginal Z’ on multiple sequential plates

Plate-by-plate Z’ trend over time

Assay signal drift — check protein or dye stability over screen duration