Positive No-Template Control in a Quantitative CMV Assay

Most likely: low-level amplicon carryover from the opened, splashed high-positive plate onto surfaces, racks, or gloves adjacent to the extraction bench, subsequently transferred into the NTC well.

Evidence for amplicon carryover:

• The NTC signal is late (Ct 37.8), consistent with a very small number of intact amplicon copies, not with a concentrated reagent contaminant.
• There is a plausible breach event (open plate outside the post-amplification area, visible splash) close in time.
• Prior runs were clean, arguing against a long-standing reagent problem.

Evidence against / must be excluded:

• A reagent or master mix contaminant would typically reproduce across plates and across multiple wells in a run, and would not correlate with a one-time bench event.
• Only one NTC was tested, so the within-run pattern is uninformative. Test 8 NTC wells using a fresh, unopened reagent aliquot and fresh tips. Reproducible positivity across those wells points to reagents; sporadic, position-associated positivity points to surface or operator contamination.
• Splash contamination can also enter through shared racks, pipettes, or the centrifuge - swab these.

The run is invalid and no patient result from it may be reported. A positive NTC violates the run acceptance criteria; the magnitude of the patient values is irrelevant to that rule.

The two low positives must be held, not released. They are exactly the results most vulnerable to a low-level contamination event: low viral loads, asymptomatic patients, and a break from six weeks of negativity. Holding them prevents a false-positive result from triggering preemptive therapy or a reduction in immunosuppression.

Actions:

• Do not report; notify the medical director and the clinical teams that results are pending.
• Re-extract the two patients from stored plasma collected before the contamination event - re-amplifying the old extract will not distinguish contamination from true viremia.
• Re-run the entire batch only after decontamination is verified.
• If the repeat is negative, the original result is discarded; if reproducibly positive at the same low level with a clean NTC, report with a comment and interpret in the context of the trend rather than a single value.

Sequence (surfaces before equipment, top to bottom):

1. Discard opened master mix, primer/probe aliquots, plate sealers, and any tube or rack present on the affected bench; use only unopened lots.
2. Wipe surfaces, pipettes, racks, and the centrifuge with 10% freshly prepared hypochlorite (amplicon DNA must be destroyed chemically), allow contact time, then wipe with water or 70% ethanol to remove bleach residue, which itself inhibits PCR.
3. Clean the plate reader and its surroundings; the splash occurred there.
4. UV the surface area overnight as an adjunct - UV is surface-only and does not penetrate tubes or dried material.
5. Decontaminate the extraction area separately, in the opposite direction of workflow (amplification to extraction), and change gloves and lab coats between zones.

Verification: 8 NTC wells across positions with fresh reagents, plus environmental swabs of the reader bench and extraction bench; swabs should be negative before patient testing resumes.

Procedural change: enforce unidirectional workflow - amplified plates are never opened in or carried through the extraction/pre-PCR area; a second plate reader or a scheduled reader time must be available so the rule is never in conflict with capacity.