Molecular Devices SpectraMax Paradigm User Manual page 47

Data Reduction
Data reduction for HTRF reads consists of two steps.
First, a ratio of the signal measured by the emission from the acceptor label at 665 nm to the
signal measured by the emission of the donor label at 616 nm is calculated and multiplied by
a factor of 10,000. This generates what is called the HTRF ratio.
In the second step, ratios are calculated that represent the relative change in the HTRF signal
compared to that of the assay background, represented by assay controls potentially named
negative or Standard 0. This relative response ratio is called the Delta F and is formatted as a
percentage, although values greater than 100 can be generated.
Data Optimization
The measurement noise is dependent on the read time per sample (time per plate or time
per well). In particular, the detection limit improves when the read time is increased.
Therefore, it is important to specify the read time when comparing measurements. For TRF,
the read time per well increases with the selected number of pulses. The time between
pulses, however, can be different on various systems.
Table 2-4: HTRF timing parameters example
Parameter
Number of pulses
Measurement delay
Integration time per cycle
(pulse)
Defining the number of flashes (pulses) cannot be used for comparative purposes because
the flash and intensity rate varies from system to system.
There are two timing parameters which can be optimized to adjust the performance of the
measurement as desired: time per plate or time per well, and integration time per cycle.
All low-light-level detection devices have some measurement noise in common. To average
out the measurement noise, optimization of the time per well involves accumulating as
many counts as possible. Within some range, you can reduce noise (CVs, detection limit) by
increasing the read time per well, as far as is acceptable from throughput and sample
stability considerations.
As the number of flashes (read time per well) is increased, several aspects of the data
improve:
Delta F values show less variability (better CVs).
Small Delta F values are better distinguished from noise.
Noise of background is reduced.
5014038 E
Value Comment
30 The number of flashes per read.
30 µs The delay to ensure the excitation pulse is no longer
detectable.
400 µs The period for accumulating the signal.
Chapter 2: Read Modes and Read Types
47