Internal Validation Of Established Procedures

In order to meet DAB/SWGDAM Guidelines for quality assurance, forensic DNA laboratories conduct the tests described above as part of the process of becoming 'validated'. These studies demonstrate that DNA typing results can be consistently and accurately obtained by the laboratory personnel involved in the testing.

Validation studies are performed with each new DNA typing system that is developed and used. For example, a lab may be validated with the PowerPlex® 1.1 kit but it would need to perform additional validation studies when expanding its capabilities to amplifying the STR loci included in the PowerPlex® 16 kit.

STR Typing Kit


AmpFlSTR Blue

SGM Plus

Profiler Plus

Profiler Plus

Profiler Plus ID

Profiler Plus, COfiler

Profiler Plus, COfiler

Profiler Plus, COfiler, Blue, Green I, PowerPlex 1.1, PowerPlex 1.2

Profiler Plus, COfiler, Green I, Profiler

Profiler Plus, COfiler

Profiler Plus, COfiler, PowerPlex 16, PowerPlex 1.1, PowerPlex 2.1

PowerPlex 1.1

PowerPlex 1.1

PowerPlex 2.1

PowerPlex 16

PowerPlex 16 BIO



genRES MPX-2

Wallin et al. (1998) Cotton et al. (2000) Frank et al. (2001) Fregeau et al. (2003) Leibelt et al. (2003) LaFountain et al. (2001) Moretti et al. (2001a) Moretti et al. (2001b)

Holt et al. (2002) Buse et al. (2003) Tomsey et al. (2001)

Micka et al. (2001) Greenspoon et al. (2001) Levedakou et al. (2002) Krenke et al. (2002) Greenspoon et al. (2004) Sinha et al. (2003a) Sinha et al. (2003b) Junge et al. (2003)

Table 16.1 Validation studies conducted using commercial STR kits.

Typical studies for an internal validation include reproducibility, precision measurements for sizing alleles, and sensitivity (e.g., 50 ng down to 20 pg) studies along with mixture analysis and non-probative casework samples. Sizing precision studies are also conducted, where the calculated allele sizes in base pairs is plotted against the size deviation from the corresponding allele in the allelic ladder with which the genotype was determined (Wallin et al. 1998, Holt et al. 2002, Krenke et al. 2002). If a high degree of precision cannot be maintained due to laboratory conditions such as temperature fluctuations, then samples may not be able to be genotyped accurately.

Each forensic laboratory develops or adopts standard operating protocols (SOPs) that give a detailed listing of all the materials required to perform an assay as well as the exact steps required to successfully complete the experiment. In addition, SOPs list critical aspects of the assay that must be monitored carefully. SOPs are followed exactly when performing forensic DNA casework.


Inter-laboratory tests are the means by which multiple laboratories compare results and demonstrate that the methods used in one's own laboratory are reproducible in another laboratory. These tests are essential to demonstrate consistency in results from multiple laboratories especially since DNA databases are now used where many laboratories contribute to the DNA profile information (see Chapter 18).

Since 1994, the European DNA Profiling Group (EDNAP) has conducted a series of inter-laboratory evaluations on various STR loci and methodologies used for analyzing them. A listing of eight published EDNAP reports that deal with STR markers may be found in Table 16.2. Each study involved the examination of 5-7 bloodstains that were distributed to multiple laboratories (usually a dozen or more) to test their ability to obtain consistent results. In all cases where simple STR loci were tested, consistent results were obtained. However, complex STR markers, such as ACTBP2 (SE33), often gave inconsistent results (Gill et al. 1994, 1998). Thus, STRs with complex repeat structures were not recommended for use in DNA databases at that time where results are submitted from multiple laboratories. However, the availability of commercial kits and allelic ladders that enable consistent amplification and typing of SE33 now mean that obtaining reproducible results is more feasible.

In the United States several inter-laboratory studies have been performed. The first large test with commercial kits was conducted by the National Institute of Standards and Technology (NIST) and involved 34 laboratories that evaluated the three STRs TH01, TPOX, and CSF1PO in a multiplex amplification format (Kline et al. 1997). This study concluded that as long as locus-specific allelic ladders were used, a variety of separation and detection methods could be used to obtain equivalent genotypes for the same samples.

As described in Chapter 5, the FBI Laboratory sponsored a STR Project from which the 13 core STR loci where chosen for inclusion in the Combined DNA Index System. A total of 22 DNA typing laboratories were involved in this project where a series of samples was systematically examined by the various laboratories. More recently, DNA quantitation and mixture studies were conducted by NIST to evaluate the differential extraction capabilities of 45 participating laboratories (Duewer et al. 2001) and quantification reproducibility with 74 laboratories (Kline et al. 2003a). These inter-laboratory studies all demonstrate that consistent results can be obtained between participating laboratories thus

Study # STR Loci Examined

Protocols Provided?

Primers/Ladders Number of Reference Provided? Laboratories Involved

1 TH01, ACTBP2


Both 14 Gill et al. (1994)

RESULT: TH01 worked well in al

l labs, ACTBP2 exhibited variable sizing with different electrophoresis systems

2 TH01, VWA,


Both 30 Kimpton et al. (199B)


RESULT: Fluorescent multiplex results were robust but problems existed with allele designations at FES/FPS and F13A1 when alternative detection methods were used

RESULT: All allele designations matched those of the originating laboratory; achieved despite variation in amplification, electrophoresis, and detection systems used

4 D21S11, FGA No Ladders only 16 Gill et al. (1997)

RESULT: Comparable results were obtained from all laboratories despite the fact that various primers and protocols were utilized; the key to standardization with more complex STR loci is to use a common allelic ladder

5 ACTBP2, APOAI1, Yes Primers and ladder 7 Gill et al. (1998) D11S554 for ACTBP2

RESULT: ACTBP2 showed good reproducibility between laboratories (<0.15 bp measured size difference); greater than expected variation with APOAI1 and D11S554 - they need locus-specific ladders

6 D12S391, D1S1656 Yes Both 7, 12 Gill et al. (1998)

RESULT: Excellent reproducibility between seven laboratories for D12S391 and 12 laboratories for D1S1656; demonstrated the need to use ┬▒0.5 bp windows centered on the appropriate allelic ladder marker

7 DYS385 Yes Both 14 Schneider et al. (1999) RESULT: Reproducible results may be obtained with a variety of separation and detection systems

8 DYS19, DYS389I/II, Yes Yes 18 Carracedo et al. (2001) DYS390, DYS393

RESULT: Reproducible results may be obtained with a pentaplex format for single source males and mixed stains containing a male component.

Table 16.2

European DNA Profiling Group (EDNAP) collaborative studies regarding DNA typing with STR markers. The purpose of these studies was to explore whether uniformity of DNA profiling results could be achieved between European laboratories.

helping support the conclusion that forensic DNA typing methods are reliable and reproducible.


Reference DNA samples are crucial to the validation of any DNA testing procedure (see Szibor et al. 2003). The National Institute of Standards and Technology, part of the U.S. Department of Commerce, is responsible for developing national and international standard reference materials (SRMs). These SRM sets are generally used to validate a laboratory's measurement capability, calibrate instrumentation, and troubleshoot protocols (Reeder 1999). Hundreds of SRMs are available from NIST but four in particular apply directly to the forensic DNA typing community. These are SRM 2390, SRM 2391b, SRM 2392-I, and SRM 2395.

The recently issued DNA Advisory Board standard 9.5 (1998) states: 'The laboratory shall check its DNA procedures annually or whenever substantial changes are made to the protocol(s) against an appropriate and available NIST standard reference material or standard traceable to a NIST standard' (see Appendix IV). A review of the various SRM materials that are now available to aid the forensic DNA typing community is given below.

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