What is the major cause of an outlier in assay results?

• A. Pipetting error (random or systematic)
• B. Calibration error
• C. Deterioration of reagent
• D. Random error

Answer: (A)

Outliers arise when one measurement is far from the others beyond what is expected from normal variability. The main driver for such an extreme value in an assay is pipetting error, because incorrect volumes of sample or reagents directly alter the concentration in that reaction and create an abnormally high or low signal. This can happen as a one-off mistake (random) or as a consistent miscalibration of technique (systematic), both of which can produce a single standout result.

Calibration error would shift results more uniformly, affecting the whole dataset rather than a single point. Deterioration of reagent tends to cause a broader drift or poorer performance across multiple wells rather than an isolated outlier. Random error is the general noise inherent in measurements, contributing to spread but not specifically explaining an extreme result by itself—the standout value is more characteristic of a pipetting mistake than just normal random variability.