What does an overflow or an “OVER” result mean?

How to prevent overflows

1. Usual root causes

When a measurement result includes the term “OVER” instead of a numerical value, the detector was oversaturated during measurement. This “overflow” is because either too much light reached the PMT (=detector) or, in the case of an absorbance measurement, the recorded value was above a certain OD limit.

2. Typical solution

• Overflow in fluorescence intensity (FI) measurements:

Overflows can be prevented by using an optimized GAIN value.
Gain is another word for amplification. Gain values should be between 60 and 220 (in some cases they can be higher, depending on the application). Gain values below 60 and above 220 will artificially reduce the instrument’s linear detection range and potentially also the performance of the assay.

Different gain settings are available:

-) Manual
-) Optimal
-) Calculated from well
-) Extended dynamic range

Tecan recommends using the Optimal gain for most fluorescence assays to prevent “OVER” results. The reader selects the gain based on the signal intensities of all wells.

A Manual gain can be used if the same samples are run multiple times and the gain was already determined. This ensures that values from different runs remain comparable, and reading time is faster because the gain does not need to be determined.

The Calculated from well option can be used if the well with the highest signal intensity is known. In this case, the reader selects the amplification factor based on the signal intensity of one selected well.

Finally, the Extended dynamic range gain is used if the signal intensities are expected to have a broad range. In this mode, two consecutive measurement runs with a high and a low gain are performed. Results from both runs are then correlated and hence comparable within one run.

• Overflow in absorbance measurements:

“OVER” results are returned if the measured OD value exceeds a certain limit. This limit depends on the reader and can be either 4.00 OD (Infinite® F50, Infinite® 200 PRO, Spark®) or 6.55 OD (Sunrise™).
Since absorbance measurement is an absolute measurement (as opposed to FI measurement), there is no setting available to lower the OD value.
To prevent “OVER” results in absorbance, dilute the affected samples.

• Overflow in luminescence measurements:

“OVER” results are returned in luminescence measurements when luminescent counts are above 10,000,000.
To prevent overflows, use attenuation filters that reduce signal intensity. Depending on the reader and its configuration, up to three different attenuation filters are available:

-) OD1
-) OD2
-) OD3 (only available in Spark® readers with enhanced luminescence module)

Luminescence signals measured with the attenuation filters OD1, OD2, or OD3 are automatically corrected by a factor of 10, 100, or 1000, respectively.

For Infinite® readers, results of luminescence measurements are always displayed in counts per second (cps). For Spark® readers, the output can be set to either Counts (readout is displayed as total counts measured in a well) or Counts/s (readout is displayed as counts per second measured in a well).

Further help

For further help during or after the installation process, please refer to our Helpdesk.

Infinite® 200 Pro How To


Link to Tecan Product Page:

Infinite® 200 PRO