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The Latest Methods Revealed

by Angelo DePalma

Forensics, the application of scientific measurement to legal or administrative questions, remains a growth market for analytical science. Although many individuals think of forensics as the analysis of crime-scene evidence, most forensic work involves routine testing of routine samples.

A 2002 Department of Justice survey of the fifty largest crime labs found that more than 85% tested for controlled substances. Other popular items subjected to forensics analysis include firearms, tools (as in the "blunt instrument" of criminal lore), DNA/biological material, fingerprints, and trace materials.

Figure 1. Miroslava Deneva, a Confirmation Technician at Forensic Laboratories, verifies vial placement on a Varian 1200L quadrupole LC/MS/MS.

Tom Gluodenis, Industry Marketing Manager for Forensics at Agilent Technologies, Inc. (Wilmington, DE), divides forensic analysis into three categories: criminalistics (trace analysis, drugs, fibers, hair, bulk pharmaceuticals, chemical accelerants), forensic toxicology (controlled substances, ballistics, poisons), and post-mortem, which can include analysis for almost any type of substance or event. Agilent's forensics analytical instrument business deals primarily with controlled substances, trace materials, fire debris, ballistics, and explosives.

Drivers of forensics instrumentation, both from the developer's/vendor's and end-users' perspectives include economics or cost per test, reliability in the lab, validity in a legal setting, ease of use, and throughput.

While gas chromatography (GC) and liquid chromatography (LC) dominate forensics science, older analytical methods such as infrared, x-ray diffraction, microscopy, ultraviolet-visible, optical emission, atomic absorption, and inductively-coupled plasma still enjoy niche applications. Not every laboratory is proficient in the use of all these instruments. Labs tend to specialize in narrow analyte or sample types, and consequently in particular instrumentation sets.

"Customers are telling us about an ever-increasing number and diversity of samples," says Gluodenis. "The pressure for greater consistency and higher throughput is continuous."

Like all analytical service laboratories, forensics labs face the dual challenges of maintaining certified personnel while keeping human resource costs down. Hence what Gluodenis terms "staffing-challenged labs." Instruments must perform powerfully, but not present such a high learning curve that users must trade off reliability, performance, and productivity. A robust data management platform, supporting high throughput plus archiving/certification activities, is essential.

It's all in the sample

James Watterson, Ph.D., a toxicologist and assistant professor of forensics at Laurentian University (Sudbury, ON), specializes in testing biological samples for pharmaceuticals, poisons, and pharmacologic performance enhancers. The latter are one of the fastest-growing specialties in forensics in the U.S. and Europe.

Figure 2. Volunteers performed hand-to-arm contact for 10 seconds followed by collection of the mixed-cell population with hydrosoluble tape. The tape was extracted using the ChargeSwitch Forensic DNA Purification Kit. (Invitrogen Corp., Carlsbad, CA) The DNA was analyzed for donor (hand) and recipient (arm) DNA. Even though the gDNA from the donor is of lower abundance than from the recipient, the identification alleles from the hand were amplified and detected. Donor alleles (from the hand) are indicated by arrows. Data provided by Petricevic, S., et. al, presented at the Fourth European-American School in Forensic Genetics and Mayo Clinic Course in Advanced Molecular Medicine, Dubrovnik, Croatia, September 5-9, 2005.

If the goal of forensics is a measurement that holds up in courtrooms and administrative proceedings, sample collection and preparation remains a top challenge. Issues related to chain of custody and sample handling can raise enough doubts for a jury to thwart an otherwise solid prosecution. All analytical techniques, be they biological (PCR, immunoassay), physical (chromatography), or destructive (elemental analysis) are subject to interference. Biological samples almost always require chemical and physical work-up to remove interfering substances before they enter the analytical workflow. Which is why solid-phase extraction, which was big news in chemical synthesis a decade ago, has become a great time-saver for forensic scientists.

Dr. Watterson relies on a gamut of analytical instrumentation: immunoassay, gas chromatography, high-performance liquid chromatography (HPLC), and chromatography/mass spectrometry (MS) combinations. MS identifies compounds by comparing mass and fragment peaks to stored or online compound libraries. HPLC/MS, an up and coming technique for forensics, has been making steady inroads into laboratories over the past decade. "These days it is not unusual for a conference to provide several days of workshops on LC/MS," says Dr. Watterson. Adoption of HPLC/MS has taken much longer in forensics than in the life sciences because the high cost for benchtop models has kept instruments out of common use. Consequently, the legal system has been slow to accept these methods as valid and admissible.

With the eventual increased acceptance of HPLC/MS comes a need for methods development and robust sample preparation methods. MS requires that samples ionize, with different ionization techniques suitable for specific types of compounds.

"The goal is to get LC/MS to the point where it is as information-dense as its GC/MS counterpart, which until recently was the gold standard for forensic analysis" Prof. Watterson states.

LC/MS is applicable to more sample types, and has greater sensitivity, than GC/MS, but the mass spectrum depends heavily on the mobile phase. "It's much more difficult to create the equivalent of NIST's 7,000-entry spectral library of drug compounds for LC/MS," Dr. Watterson observes, and transferring methods from one lab to another can be problematic.

Since samples coursing through a GC are already in the gas phase, ionization into the mass detector occurs readily and predictably. Samples emerging from an HPLC column exist as solutes, perhaps in a charge configuration unsuitable to mass analysis. GC also tends to be more reproducible, from lab to lab, instrument to instrument, or mobile phase to mobile phase, than LC methods. Agreement among different testing venues is critical for acceptance of analytical data in courtrooms.

Drugs of abuse a growth market

Testing for drugs of abuse (DOB) and ethanol has become big business, as companies in sensitive areas (transportation, construction, healthcare) seek to enforce drug-free policies. Off-site alcohol screening and testing is growing in response to court- and occupation-mandated monitoring of proscribed behavior, in this case drinking. Instead of measuring alcohol directly, as a breathalyzer does, off-site tests look for metabolites like ethyl sulfate and ethyl glucuronide, which persist metabolically up to 80 hours after alcohol ingestion.

Monitoring a convict or parolee for a panel of DOBs can cost hundreds of dollars per time point, which is why many jurisdictions use less expensive ELISA or radioimmunoassay analysis for screening and instrumental analysis to confirm the presence of a drug.

GC/MS has long been the gold standard for DOB confirmation but fails with large molecules and salts. For these, laboratories increasingly turn to LC/MS and LC/MS/MS, which Mike Gates, VP of Marketing at Forensic Laboratories (Denver, CO), calls the "platinum standard."

Figure 3. Capillary Flow Technology (Agilent Technologies, Santa Clara, CA) provides shorter chromatographic cycle times.

Forensics Laboratories, which specializes in toxicology and DOB testing, uses LC/MS/MS (tandem mass spectroscopy) to screen and confirm DOB analyses. LC/MS is still too costly for many toxicology labs; much more so LC/MS/MS. "For labs just starting up, who may do up to twenty confirmations per week, it's just not cost-effective." A THC confirmation, for example, costs around $25, and a typical instrument will set a lab back about $100,000, plus consumables, operating costs, and personnel.

Gates' lab uses a Varian 1200L LC/MS/MS quadrupole instrument, a benchtop tandem-MS system that is upgradeable and interfaces with a GC if desired. Varian, says Gates, has helped "tremendously" with methods development.

DNA: High-tech, still improving

DNA analysis, perhaps the closest thing to a buzzword in forensics, has become routine since the advent of affordable polymerase chain reaction (PCR) instruments in the mid-1990s. The challenge today is increasing sensitivity for "low copy number" samples such as fingerprints, cigarette butts, or minuscule quantities of normal DNA sources (body fluids, tissues). Low copy number samples contain less than 100 pg of DNA.

Analysis of vanishingly minuscule DNA fragments has been made possible by short tandem repeat (STR) analysis, a technique that allows identification of DNA from only a snippet of a gene three or four base pairs. According to Henrietta Margolis Nunno, Ph.D., J.D., a professor at the John Jay College of Criminal Justice (New York, NY), STR has revolutionized the capabilities of forensic DNA analysis by opening up gene amplification to mishandled or badly degraded DNA. The only drawback of the technique is that small sequences must be amplified above and beyond what is normal for PCR, which takes time and may introduce errors.

The original PCR technique, based on restriction fragment length polymorphism, requires a blood stain about the size of a quarter. STR, with samples about the size of the head of a pin, can, when pushed to its limits, amplify 10 pg of DNA the genetic contents of two human cells.

STR's sensitivity is also its principal drawback. When presented to a naïve judge or jury, a technique powerful enough to detect DNA from a handshake can easily cause a miscarriage of justice.

Despite its discriminating power STR has been slow to catch on, which is normal for revolutionary analytical technology in the staid, methods-driven world of forensics. The British justice system took about six years after the discovery of PCR to adopt the technique; in the U.S., the Federal Bureau of Investigation did not convert its gene database to STR format for nine years after other scientists had begun using the technique.

For instrumentation, Dr. Nunno has used the Applied Biosystems (Foster City, CA) real-time PCR systems, which are used in most forensics labs that handle DNA. Applied Biosystems, Promega Corp. (Madison, WI) and other companies provide kits for quantifying human DNA. She also likes Agilent's lab-on-a-chip type PCR cassette with the Agilent 2100 Bioanalyzer electrophoresis system. "Chip-based analysis doesn't give the same quality results and you can't use their data in court, but they are great for developing new separations techniques because they're fast."

Crime laboratories are beginning to adopt low to medium-throughput automated systems with pre-packaged DNA extraction chemistries. Extraction kits provide greater uniformity and inspire greater confidence in results. "There is almost no chance of human error due to pipetting mistakes, sample mix ups, or contamination from the analyst because the chemicals in the cartridges are not exposed until after the instrument door is closed," says Suzanne Kennedy, Ph.D., a product manager at Invitrogen Corp. (Carlsbad, CA).

Labs large and small are adopting benchtop instrumentation that process twelve or more samples simultaneously (the standard is six). Invitrogen's iPrep instrument, for example, handles 12 samples plus a control. Parallelism is useful even for one-off analyses since investigators usually perform tests in replicates and throw in standards and blanks.

For reference buccal swabs samples, medium-throughput benchtop instruments usually suffice for rapid, hands-free DNA prep. According to Dr. Kennedy, high-throughput, fully automated DNA extraction is available only at very large labs processing buccal swabs from felons for entry into a database, or at service labs accepting overflow from states that cannot process their own samples.