Streamlining Sexual Assault Sample Processing with the Maxwell® DE System

A Maxwell-based workflow for semi-automated differential extraction that streamlines separation and reduces manual steps in sexual assault sample processing

Margaret Ewing, Nicholas Courtney, Elizabeth Hamilton, Jonelle Thompson, Jon Drobac, and Robert McLaren, Promega Corporation

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Introduction

Differential extraction, a method used to separate epithelial and sperm cells in sexual assault evidence, has remained a key process in forensic DNA analysis since its early introduction into casework (1). In many sexual assault cases, it is an essential step in evidence processing; however, traditional manual workflows are labor-intensive and prone to user variability. These limitations can make it challenging for forensic laboratories to keep pace with rising caseloads. Manually separating non-sperm and sperm cell fractions, commonly referred to in forensic practice as Fraction 1 and Fraction 2, introduces operational bottlenecks, particularly in high-throughput environments or laboratories with limited staffing. Once the non-sperm fraction is removed, the sperm-containing pellet is washed three to five times in conventional workflows to reduce residual non-sperm DNA. Each wash involves adding buffer, resuspending the pellet, centrifuging, and carefully removing the supernatant. Although this process is time-consuming it plays a critical role in achieving clean separation. While this article uses the terms “sperm” and “non-sperm” fractions to describe the intended processing paths, these labels do not imply confirmed biological content.

The Maxwell® DE System (Catalog #AS1870) was developed to help forensic laboratories minimize the manual effort needed for differential extraction (2). This semi-automated workflow combines differential lysis chemistry with magnetic particle-based purification on the Maxwell® Instrument platform. Following lysis, the lysate is separated into two fractions: a non-sperm fraction (Fraction 1) and a sperm-containing pellet (Fraction 2). The sperm pellet is then processed in a Maxwell cartridge, where the instrument automates the wash steps that remove residual non-sperm DNA prior to elution, reducing hands-on time and supporting consistent separation (Figure 1). Designed specifically for forensic casework, the system brings increased efficiency and reproducibility to a traditionally manual and variable process.

Figure 1: Illustration of sperm fraction moving through a Maxwell® DE Cartridge. Sperm fraction is added to well 1 of the Maxwell® DE Cartridge. Cells bind to the resin, are washed, and then eluted.

In addition to reducing manual steps, the Maxwell® DE System offers flexibility in both throughput and protocol timing. When paired with the Maxwell® FSC (Catalog #AS4600) or Maxwell® RSC 48 (Catalog #AS8500) Instruments, the system can process up to 16 or 48 samples at a time, supporting high-throughput and batch-based workflows (3,4). The protocol also includes lysis incubation options for 2 hours, 4 hours, or overnight. Two-hour incubation is an option for samples collected shortly after an alleged assault or those with high sperm content. Four-hour incubation supports most casework scenarios, while overnight digestion provides added scheduling flexibility with maintained downstream performance.

The separated fractions generated by the Maxwell® DE System are directly compatible with Promega’s downstream chemistry for quantification and STR amplification, minimizing the need for additional cleanup or transfer steps. For the non-sperm fraction, users can either perform a quick 5-minute incubation at 95°C to inactivate Proteinase K or purify the sample using the Maxwell® FSC DNA IQ™ Casework Kit (Catalog #AS1550) (5). This allows laboratories to tailor the protocol to their needs, whether prioritizing speed or aligning with an established workflow.

Performance Summary

To evaluate the Maxwell® DE System, DNA quality and separation efficiency were assessed using the PowerQuant® System (Catalog #PQ5002/PQ5008), which provides key metrics including total autosomal DNA concentration, a degradation ratio ([Auto]/[Deg]) to evaluate DNA integrity, and an [Auto]/[Y] ratio to indicate the relative balance of autosomal and male-specific DNA (6). Higher [Auto]/[Deg] ratios suggest degraded DNA, while [Auto]/[Y] ratios approaching one in the sperm fraction are consistent with effective enrichment of male DNA and minimal carryover of female DNA.

Internal evaluations of the Maxwell® DE System demonstrate that it delivers reliable separation of sperm and non-sperm fractions across a range of casework-type mock samples. These results reflect effective sperm cell enrichment and optimized lysis conditions that preserve DNA integrity for downstream analysis. The following examples highlight selected performance scenarios to illustrate the system’s capability under varied sample conditions. All donor-derived samples, including post-coital and mock preparations, were collected from consenting individuals in compliance with Promega’s human subjects policy.

Differential extraction of a post-coital sample, collected five years ago approximately 15 hours after intercourse and stored at uncontrolled room temperature, was performed using the Maxwell® DE System. The sample underwent a 2-hour lysis at 56°C with Maxwell® DE lysis mix, after which the lysate was separated into sperm and non-sperm fractions. The non-sperm fraction was further incubated at 95°C for 5 minutes to inactivate Proteinase K, while the sperm fraction was processed using the standard Maxwell DE protocol. Results of quantification with the PowerQuant® System indicated 2.8ng/µl autosomal DNA in the sperm fraction, with an [Auto]/[Deg] ratio of 2.85 and an [Auto]/[Y] ratio of 1.45, indicating effective enrichment of male DNA. The non-sperm fraction yielded 21.7ng/µl total DNA with an [Auto]/[Deg] ratio of 5.85.

For each fraction, 1ng of DNA template was amplified with the PowerPlex® 35GY System (Catalog #DC3520) and analyzed on the Spectrum CE System (CE1008) (7,8).

GeneMarker®HID Software for Spectrum CE Systems (Catalog #CE3010) was used for data interpretation (9). The degradation observed in both fractions is likely attributable to the long-term, uncontrolled storage conditions of the sample. Despite this, both fractions produced interpretable STR profiles (Figure 2), demonstrating the system’s ability to perform under challenging sample conditions.

Sperm fraction

Non-sperm fraction

Figure 2: Electropherograms of sperm and non-sperm fractions from a post-coital sample processed with the Maxwell® DE System. Both fractions produced interpretable STR profiles following quantification with the PowerQuant® System and amplification with the PowerPlex® 35GY System.

While the composition of forensic samples can vary widely, internal testing shows that when uniform semen inputs are used, the Maxwell® DE System delivers consistent DNA yields from the sperm fraction.

To evaluate processing consistency, mock samples were prepared by applying 7.5µl of semen to vaginal swabs (n=8). Swabs were treated with Maxwell® DE lysis mix and incubated for 4 hours at 56°C. After incubation, samples were transferred to spin baskets, centrifuged, and the solid substrate was discarded. Supernatants (non-sperm fractions) and pellets (sperm fractions) were pooled and then divided into eight replicate samples. Quantification with the PowerQuant® System showed average DNA yields of 1.85 ± 0.30ng/µL (coefficient of variation 16%) in the sperm fraction. The average [Auto]/[Y] ratio in the sperm fraction was 1.38 ± 0.15 (coefficient of variation 11%), demonstrating effective separation and low female DNA carryover (Figure 3). These results highlight the reproducibility of the Maxwell® DE System when processing uniform samples.

Figure 3: Male [Y] concentration and [Auto]/[Y] ratios in sperm fractions from replicate mock samples processed using the Maxwell® DE System.

Conclusion

The Maxwell® DE System offers a dependable, practical, and flexible approach to differential extraction, enabling laboratories to reduce manual steps, improve consistency, and maintain compatibility with Promega’s downstream chemistry. Whether used to process a few samples or scaled for high-throughput needs, the system supports reliable separation, high-quality DNA recovery, and reduced variability, helping forensic laboratories meet the demands of sexual assault casework with confidence and greater efficiency.

References

  1. Gill, P., Jeffreys, A.J. and Werrett, D.J. (1985) Forensic application of DNA ‘fingerprints’. Nature 318, 577–9.
  2. Maxwell® DE System Technical Manual #TM734, Promega Corporation.
  3. Maxwell® RSC 48 Instrument Operating Manual #TM510, Promega Corporation.
  4. Maxwell® FSC Instrument Operating Manual #TM462, Promega Corporation.
  5. Maxwell® FSC DNA IQ™ Casework Kit Technical Manual #TM499, Promega Corporation.
  6. PowerQuant® System Technical Manual #TMD047, Promega Corporation.
  7. PowerPlex® 35GY System Technical Manual #TMD077, Promega Corporation.
  8. Spectrum CE System, 8-Capillary Technical Manual #TMD052, Promega Corporation.
  9. GeneMarker®HID Software for Spectrum CE Systems User Manual #TM555, Promega Corporation.