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The AmpFISTR^® Blue, Green I and Profiler kits are PCR kits that utilize fluorescent tags for typing STRs in nine loci and Amelogenin. The Profiler kit is a combined system that simultaneously amplifies the three loci in the AmpFISTR^® Blue kit, the four loci in the AmpFISTR^® Green I kit and three additional loci. These systems are particularly useful in forensic casework and databasing because they allow for rapid typing and automatic data analysis of several DNA loci at the same time, in addition to the advantages of STR typing.

As with any typing system used in a forensic laboratory, several tests must be run to determine the ability of the system to repeatedly produce and accurate results. The Technical Working Group on DNA Analysis Methods (TWGDAM) has set forth several guidelines that must be followed in the validation of any novel typing system as well as internal validation guidelines that each laboratory must follow before they begin casework with a particular system. The latter is referred to as Internal Validation. TWGDAM defines Internal Validation as an accumulation of test data within the laboratory to demonstrate that established methods and procedures perform as expected in the laboratory.

Validation of novel technologies requires that the locus or loci are characterized and defined, studies have been conducted on species specificity, sensitivity, stability, and mixtures, and that population distribution data has been documented. These studies are initially performed by the individual or group that has established the technology.

The North Louisiana Criminalistics Laboratory has recently completed internal validation of the AmpFISTR^® systems (using the ABI 377 DNA sequencer) for use in forensic casework. In accordance with the Quality Assurance Standards for Forensic DNA Testing Laboratories, this validation included the following requirements:

The procedure was tested using known, non-probative samples.

Reproducibility and precision of the method was thoroughly tested and monitored.

Match criteria were established based on empirical data.

Each analyst was trained and passed a qualifying exam before analyzing casework samples using the new procedure.

All material changes to the procedures as validated by ABI were documented and determined not to alter the outcome of the procedure.

In our internal validation, samples were first amplified and typed using the standard protocols provided by ABI. The quantity of DNA amplified and the number of PCR cycles were modified and established so as to provide the best results in our laboratory. The quantity of size standard and ladder to be used in the gel runs was established so as to provide peak heights that were above the minimum threshold yet below the level at which stutter becomes a problem in interpretation. The allelic ladders were run at varying concentrations and the results were used to determine the appropriate volumes required so that all three ladders can be pre-mixed and run as one combination ladder. The appropriate load volumes and acceptable denaturing times were also established.

Several reference bloods were then amplified and typed for comparison with STR results previously obtained by silver staining methods to ensure the accuracy of the typing method. One reference blood was selected for a sensitivity study, and was amplified at several quantities and was run on the ABI 377. The results were compared to those established by ABI and determined to be comparable. A similar sensitivity study was done during the control DNA provided in the amplification kit.

Precision studies were also conducted on the allelic ladder and the control DNA. Several ladders and control samples were run. An average and standard deviation was calculated for each allele. The precision data obtained in our laboratory was comparable to that provided by ABI.

The final validation studies included mixture analysis and typing of non-probative case samples. Several mixtures were prepared to include nearly all of the possible combinations of heterozygote and homozygote for each locus. The electropherograms were examined to determine the levels at which one sample is no longer detectable in the presence of the other. The results were used in writing the interpretation guidelines for the AmpFISTR^® kits.

Validation is a continuing procedure in that all kits are validated prior to use in casework. When a new lot of kits is received, one kit in the lot is tested to ensure that the reagents all perform as expected. Standard samples are amplified and typed at variable template amounts of DNA. The samples are then typed and the results compared for accuracy and precision. Once this kit passes the requirements, the entire lot is released for use in casework.