Why is there HEX amplification in my Aspergillus Multiplex Control reaction?

If a HEX amplification curve is observed in the positive control well while all No-Template Controls (NTCs) are deemed acceptable, the run should be considered valid. On some qPCR instruments, a strong FAM signal can produce a low-level HEX signal due to optical crosstalk (spectral bleed-through) between detection channels. Although the HEX fluorescence is typically weak (low RFU), it may still cross the analysis threshold and be assigned a Cq value by the instrument software. This low-level HEX signal is an optical artifact and does not represent true target amplification, contamination, or assay failure. The absence of amplification in the NTCs confirms that the reagents are free of contamination. Therefore, no rerun is required, and the results may be interpreted as normal.

Use your no-template controls (NTCs) as the referee

✅ Run is valid: If all No-Template Controls (NTCs) remain negative (no amplification in the FAM channel and a Cq > 35 on the HEX channel), a low-level HEX signal observed only in wells containing a strong FAM-positive amplification is consistent with optical crosstalk (spectral bleed-through). On some qPCR instruments, this weak HEX fluorescence may cross the analysis threshold and be assigned a Cq value despite not representing true amplification. This is an expected optical artifact and does not affect assay performance. The run may be accepted, and results reported as normal.

⚠️ Investigate before reporting: If a HEX amplification signal is observed in an NTC or in any well that is FAM-negative, the signal should not be attributed to optical crosstalk. Because crosstalk occurs only in the presence of a strong FAM signal within the same well, HEX amplification in FAM-negative wells may indicate contamination, reagent issues, or an assay setup error. In these cases, follow your laboratory's established investigation and repeat-testing procedures before reporting results.

Why This Happens

In multiplex qPCR assays, wells containing a strong FAM-positive amplification may occasionally display a weak signal in the HEX channel. This occurs because the intense FAM fluorescence can partially overlap the HEX detection window on some qPCR instruments, resulting in spectral crosstalk (optical bleed-through).

Although the HEX fluorescence is typically low (low RFU), it may still cross the analysis threshold and be assigned a Cq value by the instrument software. This does not represent true HEX target amplification.

A distinguishing characteristic of spectral crosstalk is that the weak HEX signal occurs only in wells with strong FAM amplification, and its intensity correlates with the strength of the FAM signal. When No-Template Controls (NTCs) remain negative, this is an expected optical artifact of multiplex qPCR and should not be interpreted as contamination or assay failure.

Real-Time (qPCR) Instruments will handle cross-talk in different ways

Depending on what instrument you use - you may not ever see this issue. The platforms that MGC has most commonly used behave as follows:

The Bio-Rad platforms (CFX and Opus CFX) have software that detects and ignores crosstalk by default.
The Agilent AriaMx software requires a cross-talk setting to be turned on to properly ignore crosstalk signal.
The Bio Molecular System Mic workbench software requires a decision tree in the data analysis program to be setup to ignore crosstalk.

User Guide Updates

We are updating the Aria MX data-analysis section of the product User Guide to describe this crosstalk behavior and the crosstalk correction guidance described above, so the expectation is documented alongside your workflow. This article will be kept in sync with those changes.

Why is there HEX amplification in my Aspergillus Multiplex Control reaction?

Use your no-template controls (NTCs) as the referee

Why This Happens

Real-Time (qPCR) Instruments will handle cross-talk in different ways

User Guide Updates

Further reading on spectral crosstalk in multiplex qPCR