Out-of-control aflatoxin M1 ELISA before a tanker deadline
An accredited food safety laboratory supplies a quantitative ELISA kit for aflatoxin M1 in milk, with a limit of quantification of 0.05 micrograms per kg and a regulatory limit of 0.05 micrograms per kg. Over three days the kit's calibrator optical densities fall by 30%, and the 0.05 micrograms per kg quality control sample reads 0.031, 0.028 and 0.034 micrograms per kg. A dairy client's 40 000 L silo sample is analysed in duplicate and gives 0.048 and 0.052 micrograms per kg. The client's production manager asks the analyst to report the value as 0.05 so the tanker can leave in six hours, or to re-run until a passing value appears. The quality manual requires duplicates to agree within 20% and the control to lie within plus or minus 2 standard deviations. The control chart shows seven consecutive points below the mean. The kit expires in one month and the plate reader was serviced two weeks ago.
1. Is the analytical run valid, and what should be reported?
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The run is not valid. The quality control results show a systematic negative bias of about 30 to 40%: 0.031, 0.028 and 0.034 against a target of 0.050. Seven consecutive points below the mean is a recognised control-chart rule (the 7T or 10x-type trend rule; here 7T applies), indicating a shift rather than random variation. A 30% fall in calibrator optical density points to a detection or reagent problem.
The duplicate agreement of 8% is within the 20% requirement, but that does not rescue the run because precision without trueness is misleading. The mean result of 0.050 sits exactly at the regulatory limit, and if the true bias is about minus 35%, the true concentration could be around 0.077 micrograms per kg, clearly above the limit.
Report: the result is not valid and cannot be used to declare compliance. The report should state that the analysis is invalid due to quality control failure and that a new lot and recalibration are required. It must not be rounded, averaged or presented as 0.05. Measurement uncertainty at the limit must also be considered before any compliance statement.
2. Design the technical investigation and corrective actions.
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Investigate in a defined order, starting with the most recent change: the plate reader was serviced two weeks ago. Check wavelength accuracy, filter position, linearity and background; a wavelength shift can depress all absorbance readings uniformly.
Then check the kit: storage temperature, expiry, reconstitution of calibrators, and whether the kit was shipped or stored above 8 degrees Celsius. Review incubation times and temperatures, wash efficiency, pipette calibration and water quality. Run a fresh kit lot with new controls, and run an independent reference material or proficiency test sample.
Apply the laboratory's control rules explicitly: 1-2s warning, 2-2s, 4-1s and 7T. Calculate the bias and the new mean and standard deviation, and rebuild the Levey-Jennings chart after the root cause is confirmed.
Corrective actions: quarantine the affected kit lot, raise a supplier nonconformance, retrain staff if technique is implicated, revalidate the method after any reader repair, and document the impact on previously reported results through a look-back review.
3. How should the analyst respond to the client's two requests?
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Both requests must be refused, politely but clearly, and the refusal documented. Reporting 0.05 when the measured value is 0.048 or 0.052 is fabrication: the first value would be rounded upward into a fail and the second downward into a pass, and neither is the measured result. Re-running until a passing value appears is also data manipulation, because the failed run is not discarded from the record; repeated testing with selective reporting distorts the result.
A legitimate response is to offer a valid re-test with a new kit lot and fresh calibration, run in duplicate with controls, and to explain the six-hour constraint honestly rather than certify an invalid result. The analyst should inform the quality manager, record the client's request in the file, and if pressure continues, escalate through the laboratory's impartiality procedure and the accreditation body's requirements.
The professional principle is that the laboratory's report is a legal and safety document. The tanker delay is a commercial loss; an inaccurate aflatoxin result is a public health and integrity failure.