With implications for the success of a legal case, forensic samples must be stored under optimal conditions to avoid DNA deterioration. Failure to maintain proper storage conditions can result in cases being unnecessarily discontinued by the prosecution due to inappropriate treatment of evidence.1 National guidelines and standards have been implemented that are enforced by governing bodies in forensic science, such as Interpol2 and the Federal Bureau of Investigation.3 These agencies ensure the collection, extraction, quantification, amplification, detection, and analysis of all samples in compliance with regional and/or institutional regulations, to minimize the occurrence of any contamination. In order to accurately determine a profile from a DNA sample, ensuring it is admissible in a court of law, the entire process must be documented and the protocols followed precisely, within admissible margins of error. As such, once crime scene or reference samples have been obtained, they should be aliquoted and stored as a viable library.
In order for each aliquot to be successfully and accurately identified for testing, each tube must be clearly identifiable to eliminate the chances of processing the incorrect sample. Two-dimensional bar codes are a common, effective way to clearly and uniquely identify samples. It is therefore critical that the 2-D bar codes remain readable after long-term storage, even if the sample in question has undergone regular freeze-thaw cycles or moves through chain of custody.
Sample identification and tracking
The vast majority of forensic laboratories must process a large number of DNA samples from tissue, blood, and cells. In order to streamline operations in an efficient and cost-effective manner, laboratories need to employ a robust and reliable method of sample identification and tracking. Since a great deal of forensic samples will be linked to a criminal investigation, misidentification, contamination, or degradation would have a detrimental effect on resulting data, and potentially on the outcome of the prosecution.
Forensic laboratories have to store a wide variety of evidence under conditions ranging from room temperature to cold storage (e.g., 4 °C to –80 °C), often over long periods of time. Frozen samples frequently need to be tested multiple times, and it is not uncommon to find that after multiple freeze-thaw cycles, labels can sometimes become faded or fall off. Since forensic laboratories are required to label each sample tube by hand, misidentification or duplication can become significant issues. This is especially important since lost samples can result in missing evidence, and duplication can render it useless.
Figure 1 - The incorporation of 2-D bar codes
makes each tube easily identifiable.
As a solution, 2-D bar-coded tubes (Figure 1) can be incorporated in addition to the handwritten labels to address tracking issues. Each tube is permanently tagged in order to provide the laboratory with a secure and reliable tracking method, which can be accurately monitored using spreadsheets or sophisticated LIMS. LIMS, in combination with 2-D bar-coded tubes, have the capability to tie together a vast amount of information, clearly relating each sample to a specific case. For example, when creating cuttings, extracts, or other child samples from the parent item of evidence, the 2-D bar code can be used to track each item back to the parent. The chain of custody can therefore be accurately monitored in LIMS to mitigate any potential misidentification. As such, using 2-D bar-coded tubes for laboratory sample management prevents mistakes, improves safety, and helps to streamline laboratory operations.
Due to the nature of their use, 2-D bar codes need to be hard-wearing, with the ability to maintain readability throughout the lifespan of the tube. In order to ensure that optimal quality is maintained, the data matrix bar code is permanently attached to the base of each storage tube, either via direct laser etching or encapsulation. This provides secure tube traceability, regardless of the storage system in use. The information contained within the bar code can subsequently be integrated with ease into various storage databases, enabling scientists to easily maintain a history of each item with regard to each experimental process that it has undergone.
In order to remain an effective tool, bar codes need to be able to withstand a variety of different conditions. Since it is vital that readability is maintained under typical storage conditions, the bar code must be resistant to a wide range of chemicals and abrasives as well as extremely low temperatures. The most common sample type, DNA, is typically stored long term at –80 °C, or in the vapor phase of liquid nitrogen (approximately –196 °C). However, if required for immediate experimentation and analysis, the DNA samples are often stored at –4 °C. Ideally, samples should be aliquoted and stored individually to eliminate the occurrence of multiple freeze-thaw cycles. Consequently, the risk of decreased sample quality or contamination is significantly reduced. However, due to cost or space restrictions, they are often stored in much larger batches. One way to circumvent space restrictions is to employ the use of stackable latch racks, which are designed to be stacked on top of one another for efficient use of a freezer. Individual aliquots can therefore be stored in a smaller footprint to maximize available space.
Materials and methods
Figure 2 - Two-dimensional bar-coded Thermo Scientific tubes are available in a wide range of formats and sizes.
In order to assess the ability of various tubes to maintain the readability of their 2-D data matrix bar code, three tube types were subjected to a variety of different storage conditions. Thermo Scientific Matrix, Nunc, and Abgene tubes (all registered trademarks of Thermo Fisher Scientific, Hudson, NH) (Figure 2) were exposed to the following environments: –80 °C freeze, vapor phase liquid nitrogen, and boiling water. Each tube rack used contained a column of eight tubes, filled with either 100% dimethyl sulfoxide (DMSO), distilled water, or air.
Providing a negative control, each bar code was scanned before being subjected to the various storage conditions. The tube racks were placed into either a –80 °C freezer, vapor phase liquid nitrogen, or boiling water, and subsequently allowed to thaw or cool to room temperature. A tube-thawing device was also employed, facilitating faster thawing of frozen samples. Having returned to an ambient temperature, each bar code was read before the tubes were returned to their appropriate storage conditions. Each sample underwent 10 freeze-thaw cycles at –80 °C, five cycles in vapor phase liquid nitrogen, and five cycles in boiling water.
Table 1 - Two-dimensional bar-coded tube readability results
This study demonstrates that various data matrix 2-D bar-coded tubes are able to withstand multiple freeze-thaw cycles, boiling, and cooling cycles, as well as vapor phase liquid nitrogen, without showing any signs of degradation. The results shown in Table 1 demonstrate that, regardless of content, all of the tubes from each tube rack were able to be interpreted into a numeric code after a total of 10 freeze-thaw cycles at –80 °C, five freeze-thaw cycles in vapor phase liquid nitrogen, and five boiling and cooling cycles. Therefore, freezing and thawing or boiling and cooling tubes numerous times do not affect the bar-code integrity of these three types of 2-D bar-coded tubes.
Retaining bar-code readability under common forensic storage conditions is essential for the accurate logging and tracking of various DNA samples. Due to the highly sensitive nature of the resulting data, it is pivotal that these samples maintain viability and avoid contamination. Furthermore, an accurate log of all samples facilitates easy identification, significantly reducing the chances of error to ensure that all evidence is admissible in a court of law. The use of 2-D bar-coded tubes will not only eliminate the risks of duplication and cross-contamination, but also provide clear documentation on all experimental processes that the sample has previously undergone. Therefore, scientists can accurately track and relate any child samples to the parent evidence with confidence. As a result, the use of 2-D data matrix bar-coded tubes successfully eliminates a number of potential issues that may arise within a forensic laboratory, providing a safe and reliable method of tracking, storing, and retrieving forensic samples, without impacting their credibility as evidence.
- BBC News. Police kept DNA samples with food; Aug 2009; www.News.bbc.co.uk/1/hi/England/cambridgeshire/8182707.stm.
- Interpol, International Criminal Police Organization (ICPO); www.interpol.int.
- Federal Bureau of Investigation; www.fbi.gov.
The authors are with Thermo Fisher Scientific, 22 Friars Dr., Hudson, NH 03051, U.S.A.; tel.: 800-343-0206; fax: 603-595-0106; e-mail: tal. firstname.lastname@example.org.