The following may indicate a mixture:

• The presence of more than two alleles at any one locus
• A peak in a stutter position that has a higher RFU than what would be expected, based upon the established stutter percentage for that locus
• Imbalanced and/or unexpected peak height percentages

Once the determination of a mixture has been made, it may be helpful to use the case information and/or sample type to aid in the interpretation of the sources of the mixture.

For example:

• It is common to find a mixture from touch DNA samples
• It is possible to find a mixture in a known blood sample from a deceased individual, if that person was transfused prior to death.
• It is possible to find a mixture when intimate samples are analyzed. Intimate samples are generally swabs collected from a person's body (skin, buccal, vaginal swabs)

Mixtures can vary in their complexity, and approaches should be established for how the analyst will interpret each of these categories of mixtures:

• Mixtures with major and minor contributors
• Mixtures with known contributor(s)
• Mixtures with indistinguishable contributor(s)

Many laboratories quantitatively assess the peak height or area, in an effort to determine the proportion contribution of donors to a mixture.02 More laboratories are moving towards the use of probabilistic genotyping software programs, such as STRmix or True Allele, to help with the interpretation of complex DNA mixtures.

Using percent contribution of donors in a mixture is premised on the following:

• The amplification parameters of the multiplex must be established. This can be accomplished through validation studies. Data from these studies can be used to determine peak area/peak height percentages of heterozygotes and stutter ranges for each locus. Establishing these parameters and understanding how a multiplex performs is crucial when using this mixture interpretation approach.
• The sample must be of suitable quality and quantity to ensure a balanced amplification. Degraded and low-level samples are prone to unbalanced amplification, which could prevent an analyst from reliably using this approach. Laboratories should conduct validation studies on low-level degraded samples, to include mixtures.
• The staff understands and recognizes how the multiplex behaves with these sample types.

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