Low copy number (LCN) has been a commonly used term in forensic DNA testing for much of the past decade(1)
. “LCN” associated with other words (such as DNA, testing, low template DNA) has been used in different contexts with different meanings amongst different practitioners. But what does the term “LCN” really mean? Historically, the term referred to small amounts of recovered DNA and often the augmented amplification cycles used to increase the sensitivity of short tandem repeat (STR) polymerase chain reaction (PCR) DNA-testing assays as used by some laboratories in Europe and elsewhere. During the same period of time, laboratories experimented with various procedures that increased the sensitivity of the PCR testing processes as examination and testing of evidence expanded to a wider variety of samples with limiting amounts of DNA. The addition of some of these processes alone or in combination may cause a particular sample to fall into the realm of “LCN testing” by some definitions. The existence of multiple definitions and multiple modifying words within the forensic DNA-testing community has created some confusion regarding the definition of LCN in both the forensic science and legal communities.
LCN DNA (also now termed low template DNA or LT DNA) may refer to any situation where a small amount of DNA is present in a sample based on the sample type (e.g., an item that has been handled) and/or the estimated quantity of DNA determined using routine quantification assays. Samples containing <100–200 pg of total DNA available for amplification fall into the range generally considered to be LCN DNA by most practitioners. Consequently, these samples may undergo LCN DNA-testing procedures developed to increase assay sensitivity; commonly this includes increased PCR cycles (e.g., 28 cycles increased to 31 or 34 cycles) but may include other sensitivity-enhancing techniques such as desalting or concentrating products prior to electrophoresis, reducing amplification volumes, increasing injection times during electrophoresis, using high-purity formamide, etc. More recently, LCN has been used to refer to any DNA sample or DNA profile where stochastic effects (e.g., allele and locus drop-out, heterozygous peak height imbalance and elevated stutter peaks) are likely present and/or where the alleles detected are below a laboratory-defined stochastic threshold (see accompanying article by Dr. John Butler for further discussion). Regardless of whether a quantitative-, technological- and/or qualitative profile-based definition is used, the term LCN or LT DNA generally refers to any situation where: 1) the amount of DNA available for amplification is limited due to small sample size or other factors (e.g., DNA degradation, PCR inhibition), and 2) interpreting the resulting DNA profile may require more considerations than interpreting single-source or mixed DNA profiles generated using higher amounts of DNA due to the potential for incomplete DNA profiles. Other rarely recognized definitions have arisen in court documentation (e.g., quantity of DNA on a slot blot quantitation assay, any amount of DNA below the manufacturer’s recommendation).
The courts in the United States have seen minimal challenges to DNA testing in criminal cases over the past decade, as laboratories have established routine testing procedures with few or inconsequential modifications. As a result, Frye and Daubert admissibility hearings have become a thing of the past, with attorneys and DNA analysts settling into fairly routine presentations of DNA test results and conclusions to juries and the court. However, several recent challenges to DNA testing have arisen in the United States with a focus on the definition of LCN, the procedures used and the validity of the DNA-testing results obtained. A few courts in the United States and an appellate court in the United Kingdom have ruled on the various definitions and issues of LCN DNA testing(3)
. Due to the variety of definitions being used, it is imperative that scientists carefully define the terms or use more descriptive language. This would avoid the perception that there is a lack of consensus in the scientific community and a subsequent lack of reliability, which may be a concern for the courts. In anticipation of possible future challenges it is advised that laboratories and attorneys prepare to address the scientific and legal issues regarding admissibility of evidence in criminal courts as more laboratories are testing smaller amounts of DNA from a wide variety of evidence samples.
Recommendations for the laboratory are: 1) conduct comprehensive validation studies of all techniques used in the laboratory, with particular focus on sensitivity, mixture and nonprobative-sample studies, to develop stochastic thresholds and interpretation policies that accurately reflect the data obtained and the limitations of the test system; 2) develop Standard Operating Procedures (SOP) very closely aligned with the procedures used in the validation studies; 3) report what can be defended scientifically using report wording and statistical calculations that accurately reflect the data obtained without bias; 4) make SOP, validation studies and electronic data (where printed profiles are inadequate for profile quality assessment) available in discovery; 5) provide ample training to analysts regarding validation studies, procedures and policies, and interpretation of DNA profiles with limitations prior to beginning DNA casework; and 6) use caution to not "overinterpret" the data by recognizing that some samples may have insufficient data to definitively include or exclude an individual as a possible contributor, resulting in an “inconclusive” statement.