Sperm Recovery
Written by Kirsten Kelley-Primozic   

EACH YEAR, there are more than 225,000 reported cases of sexual assault in the United States according to the U.S. Department of Justice. Of those reported cases, only 41 percent result in an arrest or are solved, meaning that many guilty offenders go free. Without validated forensic evidence, many of these cases are muddled by conflicting testimony. Many others never even make trial. Obtaining viable forensic evidence—particularly DNA evidence—in sexual-assault cases is an increasingly pressing problem for forensic laboratories across the country.

According to the National Institute of Justice, there was a backlog of about 430,000 unsolved sexual-assault cases in 2003 that involved forensic DNA analysis. Many different things tend to contribute to this tremendous backlog, including shortages of skilled forensic analysts and law-enforcement staff, limited resources for proper equipment and training, lack of suspects with whom to compare the biological evidence from the crime scene, and inefficient criminal match databases.

When a case finally reaches the laboratory and enters the processing stage, another critical bottleneck is encountered: The analysis is often slowed by outdated and time-consuming DNA isolation techniques.

As laboratories work to reduce their unprocessed cases and law-enforcement officials attempt to increase the public’s confidence in their ability to close cases, the need for methods that can rapidly provide quality DNA evidence in sexual-assault cases becomes increasingly important to everyone involved.

Overcoming the challenges
of sperm detection and collection

Sexual-assault samples are sometimes difficult and time-consuming to analyze because they may be composed of a mixture of cells and body fluids from multiple individuals. The semen in the case—specifically the sperm—that is used for DNA analysis is commingled with the victim’s epithelial cells. In certain cases the sperm are found in very low numbers in comparison with the number of epithelial cells from the victim. The challenge with these samples is to separate the sperm and the non-sperm fractions efficiently so that the non-sperm DNA does not interfere with the detection of the assailant’s DNA. However, current techniques often reduce the number of sperm recovered and limit the amount of DNA available for analysis. These present methods are also laborious and time consuming and may require expensive equipment that typical forensics labs are not always able to purchase.

One current technique:
organic preferential extraction

By far the most common technique used in modern crime labs is organic preferential extraction. This method relies on selective (i.e. preferential) lysis of epithelial cells followed by extraction of the sperm DNA using phenol and chloroform. While this technique is relatively inexpensive, the organic extraction requires manual, time-consuming work—typically eight hours to process eight samples with three hours of hands-on time.

There are now automated versions of this tried-and-true technique that can process more samples faster and produce more consistent results than manual extraction. However, both of these methods rely on separation steps that subsequently result in relatively large losses of sperm.

Another current technique:
laser microdissection

Instead of chemical separation, which results in loss of large quantities of sperm, laser microdissection (LMD) can be used to directly select and isolate targeted cells—including sperm. A portion of the sample is placed on an LMD-compatible slide and the cells are stained to identify and differentiate sperm from epithelial or blood cells. The LMD instrument then scans the slide by using the distinguishing characteristics that are manually programmed to search for sperm. The cells can then be isolated at a rate of 100 per 20 minutes, and are transferred to a micro-centrifuge cap. Finally, the collected sperm are lysed and prepared for DNA analysis.

LMD is reliable and the separation of sperm from epithelial cells is efficient. Additionally, because the sperm remains intact, a variety of DNA analysis techniques such as pyrosequencing or mitochondrial DNA sequencing can be applied. However, while the scanning of the sample is “automated,” the selection process for isolating the cells is manual.

Compared to the extraction method—which can take many hours—LMD is an ideal instrument for cutting down on time. But the expense that comes from acquiring an LMD instrument can be a major inhibiting factor for many law-enforcement laboratories.

Current trends:
micromanipulation and isolation

Microscopists at McCrone Associates—the analysis division of The McCrone Group, a materials-analysis company based in Westmont, Illinois—recently took existing techniques and methodologies that isolate small particles from a variety of matrices and adapted them in order to locate and isolate critical evidence in sexual-assault cases.

This shows the dispersal of sperm and epithelial cells (the small black clumps in the middle of the photograph) using a tungsten needle (left) and a micropipette (right).

This is an individual spermatozoon coated with water-soluble adhesive. It has been stained with Sperm Hy-Liter on the tip of a tungsten needle.

This is an image of a tungsten needle placing a spermatozoon into a drop of buffer solution in a micro-centrifuge tube so it can be sent for DNA analysis.

The technology has followed a long path of development to reach this point. In the early 1960s, Anna Teetsov of McCrone Associates developed techniques to pick small particles from airborne particles collected on the filters from power-plant stacks and, more recently, foreign particles in histological tissue specimens from hip replacements. An adaption of these same micro-manipulation and isolation techniques have been demonstrated to be extremely useful in rapidly segregating low numbers of sperm (less than 100) in sexual-assault cases.

Using Kernechtrot-Picroindigocar-mine (KPIC) stained smears—or smears stained with Independent Forensics’ Sperm Hy-Liter—while employing fluorescence microscopy, analysts can efficiently locate sperm in the sample. After coating the slide with a water-soluble adhesive, analysts can isolate (or “pick”) the sperm from the slide using a compound microscope. The sperm can then be processed for DNA analysis.

The isolation of each sperm requires only seconds, and the entire process to prepare individual sperm takes less than ten minutes, making it the most efficient method currently available. With the sperm cleanly isolated, sensitive DNA analysis methods—such as mitochondrial or pyrosequencing, which require intact sperm—can then be used to characterize the sperm DNA. Currently, laboratories such as Midwest Research Institute and Mitotyping Technologies are working with McCrone Associates to determine if such techniques can be used even in the case of a single isolated sperm, which would make almost any sexual-assault case available for DNA investigation and increase the likelihood of convictions in sexual-assault cases.

The sperm micromanipulation and isolation technique requires relatively inexpensive microscopes that most labs already possess and supplies they can purchase for less than $100. All that is required is some training. The technique is taught at The McCrone Group’s College of Microscopy as part of a course identified as “COM 700: Body Fluid Identification and Microscopic Methods of Sperm Detection for For-ensic DNA/Serology/Biology”.

Tuition and expenses for the class can be waived for candidates from state and local crime laboratories that qualify for a National Institute of Justice (NIJ) grant recently awarded to the College of Microscopy. It only takes two hours to learn the micromanipulation and isolation technique. That classtime includes hands-on practice and technique development.

How to get more information
about the course:

To learn more about “COM 700: Body Fluid Identification and Microscopic Methods of Sperm Detection for Forensic DNA/Serology/Biology” at McCrone Group’s College of Microscopy, go to: www.collegeofmicroscopy.com/spermdetection

About the Author

Kirsten Kelley-Primozic is a research microscopist at McCrone Associates, Inc. She adopted techniques developed at McCrone Associates and applied them to the micromanipulation and isolation of sperm technique. Primozic teaches at McCrone Group’s College of Microscopy. She can be reached at: This e-mail address is being protected from spam bots, you need JavaScript enabled to view it

"Sperm Recovery," written by Kirsten Kelley-Primozic
November-December 2008 (Volume 6, Number 6)
Evidence Technology Magazine
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