High Coefficient of Variation at Low Standard Concentrations

Symptom

Replicate wells at the bottom of the standard curve (low analyte, high OD) show high CVs >15-20%, while mid-curve and high-concentration points demonstrate acceptable reproducibility.

1. 1 Higher OD values inherently more susceptible to pipetting and reader variability
2. 2 Minimal competitive displacement at low concentrations amplifies measurement variance
3. 3 Sample concentrations falling outside optimal middle range of standard curve
4. 4 Edge effects on microplate causing differential evaporation or temperature gradients

1. 1 Shift standard curve concentration range upward to move samples into mid-curve range with lower CVs
2. 2 Dilute samples less (e.g., 1:2 instead of 1:5) to increase measured concentration into optimal range
3. 3 Use calibrated multichannel pipettes and increase number of replicates from duplicates to triplicates
4. 4 Avoid using outer edge wells of plate or include blank rows/columns as thermal buffer zones

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