When the quantity of DNA being analyzed is very low, it may be difficult to distinguish true low-level peaks from technical artifacts, including noise. Consequently, most forensic laboratories have established peak-height thresholds for "scoring" alleles. Only if the peak-height, expressed in relative fluorescence unit (RFU), exceeds a standard value will it be accepted.
There are no firm rules for establishing threshold values. Each laboratory must set its own as part of its validation procedure. The threshold may be determined experimentally on the basis of observed signal-to-noise ratios, or may be arbitrarily set to a level established by manufacturers or published data. Applied Biosystems, Inc. (ABI), which sells the most widely used systems for STR typing, has recommended a peak-height threshold of 150 RFU, saying that peaks below this level must be interpreted with caution. However, many crime laboratories that use the ABI system have set lower thresholds based on their own studies, typically 50 to 100 RFU.
The lower threshold is a measure of the sensitivity of the procedure. Most laboratories establish both lower and upper thresholds for data interpretation, thereby establishing a window to interpret data.
A laboratory’s threshold can be influenced by a variety of factors. For example, there are sensitivity differences between the types of instrumentation (e.g. capillary electrophoresis (CE) instruments and slab gel instruments) and within any one type of instrument (e.g. between different ABI 310 instruments).
Many laboratories have noted the varying sensitivities of capillary electrophoresis (CE) instruments, which tend to be more sensitive and have better resolution than gel-based systems. Some laboratories have established thresholds within their laboratory that vary depending on the sensitivity of the specific instrument.
The upper threshold is crucial when reviewing data from high quantity DNA samples. Samples with high quantities of amplified DNA will have high RFU values that can oversaturate the instrument’s ability to detect the sample. This can lead to difficulty in interpretation because an accurate measurement with respect to the peak heights and/or areas may not be obtained. This can be especially problematic when working with mixed samples.
The instrument’s software analyzes and makes sizing determinations based on the lower threshold established by the laboratory. Some laboratories have adopted a procedure to evaluate data below their RFU threshold in an attempt to interpret data for exclusionary purposes. For instance, a laboratory can have a lower threshold at 100 RFU. If additional data is detected below that threshold (e.g. 50 to 99 RFU), it could be evaluated for exclusionary purposes.
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