If standards are improperly prepared, contaminated, degraded or pipetted inconsistently, the standard curve becomes unreliable - leading to inaccurate quantification of your samples.

Missing or poorly implemented NSB/S0 (zero standard) controls let background noise be mistaken for true signal, reducing specificity and increasing false positives.

Without a positive control (a sample with a known high signal) it is hard to tell a true negative from a failed run caused by, for example, reagent degradation.

Inconsistent placement or omission of spiked-matrix or QC samples causes variability between runs and plates, making data hard to compare or validate.

Omitting a spiked-matrix control can mask matrix effects, causing over- or under-estimation of the analyte due to interference from serum, plasma or tissue lysates.

Improper pipetting or reusing tips between standard wells causes cross-contamination that distorts the standard curve and downstream quantification.

Without wavelength reference wells, plate-reader measurements can include background absorbance from plastic, buffer or media, skewing the data.

Expired or badly stored control reagents give reduced signal or complete assay failure that is hard to interpret without reliable positive and negative controls.

If blanks or negative controls are missing or mislabelled, it is difficult to set accurate detection thresholds, causing false positives or negatives.