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Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell linesduplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at !80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

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Anonymous liquid blood samples with self-identified ethnicities were purchased from Interstate Blood Bank (Memphis, TN) and Millennium Biotech, Inc. (Ft. Lauderdale, FL) and extracted using a modified salt out procedure (1). The extracted DNA was then quantified using UV spectrophotometry at 260 nm and a PicoGreen assay (2). A 150-µL aliquot of the extracted DNA solution was directly quantified in a Cary 100 double-beam spectrophotometer (Varian Analytical Instruments, Walnut Creek, CA). Low volume micro-cuvettes allowed for accurate absorbance measurements (A = 0.2 to 0.6) without prior dilution of the stock extracted DNA. Sample concentrations were adjusted to 1 ng/µL for typing purposes using the PicoGreen assay values. Fifteen autosomal STR markers (the 13 CODIS core loci and D19S433 and D2S1338) were typed along with amelogenin using the Applied Biosystems AmpF1STR® Identifiler™ kit (3). PCR amplification was carried out on a GeneAmp® 9700 (Applied Biosystems) using 1 ng of DNA according to kit protocols (3) with the exception of reduced volume reactions (5 µL instead of 25 µL) and reduced cycles (26 instead of 28). Amplification products were diluted 1:15 in HiDi™ formamide and GS500-LIZ internal size standard (Applied Biosystems) and analyzed on the 16-capillary ABI Prism® 3100 Genetic Analyzer without prior denaturation of samples. POP™-6 (Applied Biosystems) rather than POP™-4 was utilized for higher resolution separations on a 36 cm array. Samples were injected