Troubleshooting / Common Issues
LoQ cannot be determined — all CV% values are below the threshold
Possible causes
- –The method performs with acceptable precision throughout the entire tested concentration range — the LoQ lies below the lowest tested level
- –The CV% threshold is set too high relative to the method's actual precision capability
- –Concentration levels do not extend low enough to capture where CV% rises above the threshold
Recommended action
This is the most common warning. It does not necessarily indicate a problem — it may mean the method is performing well. To resolve it: (1) test additional concentration levels at lower concentrations to find where CV% rises above the threshold, or (2) consider whether the CV% threshold should be reduced to better reflect the actual precision requirements of the clinical application. Review the CV% plot to confirm the fitted curve is trending upward at the lowest tested level — if it is flat or trending downward, lower concentration levels are needed.
LoQ cannot be determined — all CV% values are above the threshold
Possible causes
- –The method has poor precision across the entire tested range — the LoQ lies above all tested concentrations
- –CV% threshold is set too low (too strict) for the method's inherent imprecision
- –Concentration levels do not extend high enough to reach the region where CV% falls below the threshold
Recommended action
Test additional concentration levels at higher concentrations where CV% is expected to decrease. If CV% remains above the threshold even at concentrations well above the expected LoQ, investigate the method's fundamental precision performance — review calibration, reagent condition, and instrument maintenance records. If the threshold is intentionally strict, verify that the acceptance criterion is appropriate for the clinical use case.
Large discrepancy between Measured CV% and Fitted CV% at one or more levels
Possible causes
- –Outlying measurements at that concentration level — a small number of replicates may disproportionately inflate the SD
- –The power model may not be a perfect fit for this analyte's precision profile across the full range
- –Sample preparation variability at that specific level
Recommended action
Review the raw replicate data at the discrepant level in the concentration level table. Check for individual outlying results that may be inflating the SD and measured CV%. If a valid reason for exclusion exists (documented instrument error, sample handling issue), the data point may be removed with justification. If the model fit is systematically poor, consider whether the concentration levels span an appropriate range for the power model assumption.
Large difference in LoQ between reagent lots
Possible causes
- –Genuine lot-to-lot variability in precision performance at low concentrations
- –One lot stored or handled differently, causing precision degradation
- –Insufficient replicates per lot to obtain a stable CV% estimate
Recommended action
Review per-lot statistics and the CV% plot for each lot to identify which lot is driving the higher LoQ. Investigate that lot's storage conditions and handling history. Testing additional lots can help determine whether the outlying lot is anomalous or whether variability is inherent to the reagent system.