From Backlog to Breakthrough: Expediting SAEK Processing with Automation, Y-Screening and QA

Anna Bennett, PhD, Promega Corporation

The forensic science community is grappling with an overwhelming backlog of sexual assault evidence kits (SAEKs), a crisis that not only delays justice for survivors but also strains the capacity of forensic laboratories. Addressing this challenge requires a multifaceted approach that enhances efficiency, reduces human error, and ensures the reliability of DNA analysis. This article explores three critical components that together form a comprehensive strategy to tackle the SAEK backlog: implementation of Y-screening techniques, adoption of automation in forensic processes, and adherence to stringent quality assurance (QA) standards.

Together, these advancements offer a pathway to not only reduce the current backlog but also prevent future delays, ensuring that forensic labs can provide timely and accurate results in the pursuit of justice.

Forensic Y-Screening for Ending the Rape Kit Backlog

The backlog of SAEKs in crime laboratories gained national attention when a group of journalists uncovered the issue in a search of crime lab records in 2015. Reasons for the massive kit backlog include funding, time and decision not to prosecute the case. As a result, survivors are left without answers for years.

Serological and autosomal single-tandem repeat (STR) testing is the conventional process for testing sexual assault samples in forensic science. However, both methods are time-consuming, laborious and offer limited sensitivity. Y-Screening, in contrast, is a process that is faster, cheaper and more sensitive than autosomal screening. This method can help labs process their SAEK backlog more efficiently and solve more cases.

The Backlog Problem

The SAEK backlog exists in two places: 1) in evidence storage facilities where a SAEK is booked into evidence, but DNA analysis has not yet been requested and 2) in crime labs where tests await DNA analysis. Lack of resources is cited as one of the major reasons behind the SAEK backlog. On average, testing a kit costs between $1,000–1,500 (US). Lack of funding combined with increased workload has strained laboratory budgets and contributed to the increased backlog. Similarly, police departments lack the personnel needed for shipping untested kits to labs, tracking submitted kits and following up on leads that result from the SAEK testing.

RAINN (the Rape, Abuse, & Incest National Network) has dedicated several initiatives in the form of grants toward decreasing the backlog of sexual assault samples in public labs. But an article published on the RAINN website addressing this problem states, “It remains imperative to ensure that public crime labs have the necessary capacity to keep up with testing demands.” While the backlog is steadily dwindling, improving efficiency and efficacy of kit testing is still in high need in public forensic labs.

Limitations of Current Testing Methods

Serological screening is the examination of bodily fluids lifted from a SAEK. The goal is to determine if there is any evidence of bodily fluids or contact and to identify potential sources of DNA for further analysis. Traditional serology screening methods may involve microscopic examination, acid phosphatase testing, microscopic sperm detection or other techniques that detect bodily fluids. Serological screening alone was commonly used before the advent of DNA analysis and has several limitations.

Serological tests are limited in both sensitivity and specificity. They fail to detect small amounts of a substance, which results in an indefinable sample. Furthermore, serological screening methods (e.g., microscopic examination) can provide information about the presence of specific bodily fluids. However, they may not offer detailed information about individual characteristics within those fluids, such as DNA profiles or specific antigens. Additionally, some bodily fluids, such as semen, saliva, or certain types of blood stains, may degrade over time, making it more challenging to detect and analyze them accurately. The longer the sample has been exposed to environmental conditions, the higher the likelihood of degradation, which can affect the reliability of serological screening results and produce false negatives.

Given these limitations, serology alone cannot be used in identification of genetic contributions to bodily fluids in a SAEK sample. Therefore, DNA based approaches following serology have been the benchmark for forensic analysis of SAEK. Specifically, single-tandem repeat (STR) testing is the gold standard for testing samples containing human DNA. In 1997, the Federal Bureau of Investigation (FBI) identified 13 autosomal STR loci to establish the core of the Combined DNA Index System (CODIS). This database includes profiles contributed by federal, state and local forensic laboratories. The core set of CODIS STR loci are widely used today in both forensic identification in property crimes and sexual assault cases.

During standard STR testing, specific regions of the autosomal DNA are amplified and analyzed. The number of repeats at each STR locus varies between individuals, making them useful for genetic profiling and individual identification. However, this process is labor intensive (extraction, quantification, amplification, capillary electrophoresis, data analysis, and profile interpretation of large amounts of DNA on low-concentration samples) and often ineffective when samples are aged or improperly stored due to breakdown of enzymes used in initial serological screening. Given the large backlog of samples that span decades, autosomal testing is insufficient to solve the backlog problem.

Y-Screening is More Efficient

Y-Screening refers to the analysis and examination of the Y-chromosome, or the male sex chromosome, in DNA samples. Y-screening is primarily used in cases where the DNA sample is limited, degraded or mixed with DNA from multiple individuals – which is the case for most of the SAEK backlog. By targeting specific regions of the Y-chromosome, forensic scientists can amplify and analyze the Y-STR profile. Additionally, conventional screening results offer analysts little predictive power as to what DNA profile may result. Recognizing the limitations and challenges associated with traditional serology methods, new  SWGDAM recommendations are now instructing the use of a “Direct to DNA” approach where DNA analysis – particularly Y-screening – performed before serology to maximize the chances of obtaining CODIS eligible profiles, which is even more efficient than pervious pipelines.

Y-screening in forensic science has alleviated the limitations of serology and traditional STR screening by targeting the male Y chromosome directly from sample, eliminating the need for time-consuming serological testing combined with autosomal screening. Y-Screening is a process that amplifies STR regions on the Y chromosome directly from sample (“direct-to-DNA") and the resulting Y-STR profile is unmasked in the presence of female DNA. Furthermore, Y-STR testing is more sensitive: Y-STR profiles have been recovered in cases where bodily fluids were first undetected by autosomal STR or other serology means. Thus, Y-STR analysis can reinvigorate the investigation of kit backlogs that have screened negative or produced only the victim's DNA.

The Role of Automation in Reducing the SAEK Backlog

The forensic science community faces a significant challenge with the backlog of SAEKs. Delays in processing these kits stem, in part, from the complexity of extracting and analyzing degraded and mixed DNA samples.

Here we explore the role of Y-Screening for prioritizing valuable samples and discuss the drawbacks of manual DNA extraction—which is time-consuming and prone to human error. We then delve into how automation can revolutionize forensic laboratories by increasing efficiency, reducing variability and improving the quality of DNA analysis. We also discuss the efficacy of one automated platform (the Maxwell® RSC 48 Instrument) in handling forensic samples to underscore the benefits of adopting automated systems in forensic analysis.

Navigating Challenges in Forensic Analysis

The backlog of SAEKs creates a cascade of problems within the justice system. Several factors contribute to delays in SAEK analyses, many centered around capturing genetic information from samples. Often, evidence collected from crime scenes consists of mixed or degraded DNA samples, complicating analyses.

In these cases, Y-Screening is often used to isolate male-specific DNA, so that forensic labs can prioritize samples more likely to yield probative genetic data. Once the Y-STR profile—or any DNA profile—is extracted from a sample, this genetic information is compared to local samples or searched more broadly through databases like the Combined DNA Index System (CODIS). Y-screening streamlines forensic analysis and potentially accelerates the resolution of cases.

Manual DNA extraction in forensic science is a resource-intensive process that must adhere to strict standard operating procedures for evidence to be admissible in court. The initial steps in manual extraction involve preparing sensitive samples—such as tissues, buccal swabs or fluids—which require careful handling to prevent contamination. Then, each sample undergoes a series of pipetting and centrifugation steps to achieve cell lysis, isolation and washing. Laboratory personnel must closely monitor, measure and adjust reagents and conditions based on the sample’s response to treatment. Each of these steps takes significant time and attention, requiring meticulous adherence to protocols to ensure the integrity and purity of the DNA for subsequent analysis.

The meticulous and time-consuming nature of manual DNA extraction exacerbates other challenges related to funding and personnel shortages, further compounding forensic labs’ SAEK backlog issue.

Automation Speeds Up Analysis While Minimizing Errors

Automation enables forensic scientists to process hundreds of samples simultaneously with speed, efficiency and flexibility. It fundamentally transforms the capacity of forensic laboratories to process samples at scale. Furthermore, as forensic laboratories face fluctuating caseloads and complex samples, automation allows labs to efficiently adjust to changes in demand without proportionally increasing staffing. Many automated systems are also equipped to handle a diverse array of sample types and testing requirements. Increased throughput and flexibility are crucial for addressing the SAEK backlog problem and preventing future backlogs.

Manual DNA extraction introduces human error through variables like improper sample handling, which can lead to poor quality or cross-contamination. Automation minimizes this variability by executing predefined protocols so that every sample is treated consistently. This standardization is crucial for forensic analysis, as consistent results are necessary for evidence to be admissible in court.

While the upfront costs of automation may seem like a barrier, the long-term savings can make automation a judicious investment for an environment where budgets are constantly fluctuating. Automated systems significantly reduce the labor-intensive aspects of DNA extraction and processing, lowering operational costs and minimizing the likelihood of costly errors and subsequent re-analyses.

In short, automation streamlines tedious workflows, decreases sample variability, and a large initial investment pays dividends in the future.

Validating an Automated System for DNA Extraction from a Variety of Sample Types

A range of instrument options for automated DNA extraction are currently available. We carried out a series of validation studies testing the performance of the Maxwell® RSC 48 Instrument used in conjunction with the Maxwell® FSC DNA IQ™ Casework Kit. This system provides medium- to high-throughput genomic DNA extraction from common forensic samples.

The Maxwell® Rapid Sample Concentrator 48 (RSC 48) Instrument uses an automated workflow to purify DNA from preprocessed forensic samples. It operates in conjunction with the Maxwell® FSC DNA IQ™ Casework Kit, which uses paramagnetic particles to prepare samples for STR analysis. The validation tests covered a range of sample types including human blood, saliva, buccal swabs and semen. Key performance metrics evaluated included yield, purity, and integrity of the extracted DNA. Additionally, the studies assessed the Maxwell® system’s ability to maintain consistency across multiple runs and different types of samples. The efficacy of DNA extraction in the presence of common forensic inhibitors was also tested, confirming robustness in real-world forensic conditions.

The validation studies confirmed that the Maxwell® RSC 48 Instrument meets or exceeds all required standards for forensic DNA extraction and purification. The results demonstrated that the instrument provides high-quality, reliable DNA yields, which are essential for accurate STR profiling in forensic science. The use of an automated system significantly reduces the potential for human error, ensures reproducibility of results and speeds up the processing time.

Final Thoughts and Future Directions

Adopting automated DNA extraction systems is crucial for forensic labs as it offers a solution to resolve the pressing backlog of sexual assault evidence kits. By automating extraction, forensic laboratories can handle larger volumes of samples with greater accuracy and less human error, leading to faster and more reliable results. Automation is poised to resolve cases more efficiently and improve the overall integrity of forensic evidence. As forensic technology evolves, embracing automation will be key to addressing current challenges and ensuring that survivors of sexual assault receive timely outcomes. The continued validation and integration of such technologies will be crucial in shaping the future of forensic analysis and in reinforcing our justice systems.

How to Meet Quality Assurance Standards in DNA Analysis

Quality assurance (QA) is an integral aspect of forensic DNA testing as it ensures accuracy, reproducibility, sensitivity and reliability in the results obtained from analyzing DNA. Rigorous standards set by entities such as the Federal Bureau of Investigation (FBI) or the European Network of Forensic Science Institutes (ENFSI) must be met to ensure a level of trust in forensic data. Here, we define quality assurance standards and validation procedures and provide an example of a tool designed to support forensic labs in meeting quality assurance requirements.

DNA extraction, amplification, analysis and STR profiling all take place within the forensic lab and must conform to quality standards. The production of precise and reliable DNA analyses begins with lab equipment that has been validated for compliance with all the appropriate quality controls. Instrument validation and quality control measures ensure that a forensic lab’s procedures are in keeping with the most up-to-date methods and technologies in order to maintain the scientific validity of their analyses over time. These efforts provide DNA analysts greater confidence in the data informing their analyses and that their findings are acceptable evidence.

To meet the stringent FBI QAS, a DNA analysis system in a forensic lab would need to demonstrate the following:

Instrument Validation and Quality Control: Validation involves a comprehensive assessment to ensure that a DNA analysis system consistently produces accurate and dependable results that are admissible in a court of law. It includes testing the system with known samples to confirm its reliability. Quality control measures should be in place to monitor and maintain the system's performance. These include routine checks, calibration and regular maintenance to detect and rectify any deviations from established standards.
Data Accuracy and Precision: The production of accurate and consistent DNA profiles is crucial to maintaining a forensic lab’s credibility. The system should generate results with minimal variation between tests and different analysts. Data interpretation, allele calling and peak sizing should be reliable and reproducible to minimize errors and ensure consistency in results.
Documentation: Comprehensive documentation is essential for transparency and traceability. It should cover all aspects of the system's processes, protocols, validations and quality control measures. Detailed records facilitate auditing and provide evidence of compliance with established standards and procedures.
Proficiency Testing: Participation in proficiency testing programs is a critical element in quality assurance. These programs involve analyzing blind samples provided by external agencies to assess the accuracy and performance of the system. Proficiency testing helps ensure that the DNA analysis system consistently produces reliable results and is on par with other accredited laboratories.
Training and Competency: Personnel operating the DNA analysis system should receive comprehensive training to ensure they are proficient in using the technology and interpreting results accurately. Demonstrated competency is necessary to minimize errors and maintain the quality and reliability of the analysis.
Chain of Custody: Maintaining the chain of custody for DNA samples preserves their integrity and admissibility as evidence in legal proceedings. The system should have well-defined protocols for handling, documenting and securing samples from the moment they are collected to their final analysis, ensuring they are not contaminated or tampered with.
Reporting: Proper legal and investigative procedures require clear, accurate, and comprehensive reporting of DNA analysis. Reports should include detailed information on the methodology used, results and any relevant statistical data, making them suitable for courtroom presentation and supporting investigative efforts.

By adhering to these quality assurance requirements, forensic DNA analysis laboratories can maintain the highest standards of accuracy, reliability and integrity in their work.

You can read about a capillary electrophoresis instrument proven to meet QA standards set by the FBI in this white paper. The study includes baseline noise and threshold evaluation, precision, resolution, accuracy, reproducibility, sensitivity, signal variability, dye balance, spectral artifact analysis, contamination assessment, mixture samples, and direct amplification. The results of the study demonstrate the capabilities of the instrument, its suitability for use in a forensic casework or database laboratory and detail the level of rigorous testing necessary to ensure production of reliable forensic DNA tools.