Forensic DNA contamination

Part IV in a series on the Knox/Sollecito case

Before we examine the bra clasp in the murder of Meredith Kercher, we need to explore the problems of forensic DNA contamination. This background may also help to explain the problems relating to the DNA sample culled from a kitchen knife discussed in part I of this series.

Definition

In An Introduction to Forensic DNA Analysis, p. 14, Norah Rudin and Keith Inman “define contamination as the inadvertent addition of an individual’s physiological material or DNA during or after collection of the sample as evidence…A contaminated sample is one in which the material was deposited during collection, preservation, handling, or analysis.” When this is done deliberately, this constitutes evidence-tampering, although there is no bright line between tampering and contamination.

Proper collection of evidence

Dick Warrington has written two useful articles on contamination for Forensics magazine. He stresses the importance of changing gloves frequently and using disposable tools. If disposable tools are not available, one must clean them between handling different pieces of evidence. In the 2009 article he cautions, “If you pick up one piece of evidence and then pick up another piece of evidence you can transfer evidence from the first item to the second item. You can avoid this kind of cross-contamination if you remember to change your gloves before handling each piece of evidence.”

Sources of contamination

The polymerase chain reaction (PCR) technique is used to increase the amount of (amplify) DNA by a maximum of 2ⁿ times, where n is the number of cycles of PCR. Because n is typically greater than twenty, this process amplifies the amount of DNA in every sample by over one-millionfold.

In Forensic DNA Typing, p. 152, John M. Butler notes “Contamination implies the accidental transfer of DNA. There are three potential sources of contamination when performing PCR: sample contamination with genomic DNA from the environment, contamination from samples during preparation, and contamination of a sample with amplified DNA from a previous PCR reaction (Lygo et al. 1994). The first source of contamination is largely dependent on sample collection at the crime scene and the care taken there by the evidence collection team (see Chapter 3). Environment contamination can be monitored only in a limited sense by ‘substrate controls’ (Gill 1997). The latter two sources of contamination can be controlled and even eliminated by using appropriate laboratory procedures and designated work areas (see Chapter 4).”

Footnotes for Butler

Gill, P. (1997) Forensic Science International, 85, 105-111.

Gill, P. and Kirkham, A. (2004) Journal of Forensic Sciences, 49(3), 485-491.

Lygo, J.E. et al., (1994) International Journal of Legal Medicine, 107, 77-89.

Dr. Donald Riley writes, “It is often said that the most critical source of PCR contamination is DNA from previous PCRs. Again, a PCR produces many DNA copies of the target DNA sequences. Due to shear number, these copies (called amplicons) are a hazard for future PCRs… However, a more dangerous source of contamination is what is called genomic DNA. This is DNA that hasn't yet been amplified. Genomic DNA doesn't have the high concentration of the target DNA copies but is a hazard because genomic DNA could produce an entirely false DNA profile. Full profile contaminants have been documented on multiple occasions and in multiple laboratories. Partial profile contaminants are more common and sometimes constitute a poorly recognized risk in using partial profiles in evidentiary samples as evidence. When contamination occurs there is rarely any way to confirm how it happened.” (emphasis added) The wonder of the PCR technique, its ability to make almost unlimited quantities of DNA, is also its danger.

Preventing and detecting contamination in the lab: the limits of negative controls

Donald Riley delineates some parallels between sterile technique and the techniques used in DNA forensics lab to minimize contamination. However, he also notes that clinically sterile equipment or reagents might still harbor DNA and that DNA samples lack an immune system to ward off contaminants. Moreover, good technique does not ensure a zero probability of contamination. Hence, there is a need for negative control experiments.

Terri Sundquist and Joseph Bessetti of Promega Corporation write:

“A ‘reagent blank’ control consists of all reagents used during sample processing but contains no sample. This control is used to detect DNA contamination of the analytical reagents used to prepare the sample for analysis. In a separate negative control reaction, water is used instead of extracted sample or reagent blank. This negative control reaction is often referred to as the ‘no-template’ control and allows identification of contamination in the amplification reagents themselves.”

However, Donald Riley noted some deficiencies in negative controls, “Alternatively, the blank may show no profile, consistent with, but not proving that contamination didn't occur. Unfortunately, a few forensic DNA laboratories omit their controls…Good PCR technique is no guarantee that contamination didn't influence the results. Steps must be taken to try and detect contamination. Negative controls are blank PCRs that have all the components of the evidentiary PCRs but have no other DNA added intentionally. Fortunately, there are often two negative controls used, one when the DNA is extracted, and another when the PCR is set up. Any PCR signal in the negative control would warn that contamination has occurred. Unfortunately, the negative controls are virtually the only warning of PCR contamination. Negative controls may alert the analyst to general contamination occurring within the lab or the lab reagents. These controls don't offer protection against contamination occurring before the samples arrived at the PCR lab. Negative controls also can't rule out contamination of individual samples. The individual samples lack individual signs of contamination if it occurs. Unlike a human patient, a PCR is incapable of showing signs of infection (contamination) such as fever or undue pain. PCRs also have no immune system to ward off contaminants.”

Butler, pp. 152-154, implicitly recognizes the problems associated with the PCR amplification process:

“The possibility of laboratory contamination is assessed with ‘negative controls’ that test for contamination of PCR reagents and tubes. Basically, a negative control involves running a blank sample through the entire process in parallel with the forensic case evidence. The same volume of purified water as DNA template in the other samples is added to a negative control PCR reaction. If any detectable PCR products are observed in the negative control, then sources of contamination should be sought out and eliminated before proceeding further…To reduce contamination problems during the laboratory examination of DNA samples, all pre-PCR and post-PCR amplication reactions should be kept physically separate. Laboratory contamination is probably impossible to avoid completely, but can be proactively assessed with negative controls and staff elimination databases (Gill and Kirkham, 2004).”

Norah Rudin and Keith Inman (p. 15) state: “Once the sample is in the laboratory where it can be dried and chilled, the potential for contamination is mostly from other samples undergoing processing at the same time…Precautions include processing evidence and reference samples separately in space and time, restricting PCR product to an isolated room, and using controls to detect contamination in any batch of samples.” (emphasis added)

But William C. Thompson is clear about the limitations of negative controls,

“A false incrimination can also occur through cross-contamination among evidentiary samples. Even labs that are careful to test reference samples separately from evidentiary samples often process all of the evidentiary samples from a case together, creating the potential for false matches. I recently reviewed a case processed by the Los Angeles Police Department DNA laboratory in which samples from a bloody murder scene were being processed in the same batch as samples from items collected in a suspect’s house. Due to an analyst’s error in the case, DNA from the murder victim accidentally ended up in a control sample. This error was detected because the control sample was a “blank” which was supposed to contain no DNA. However, it was merely happenstance that the accidental transfer of DNA ended up in a blank control rather than another sample in the same batch. If the victim’s DNA had instead ended up in one of the samples from the suspect’s house, I believe that the error would not have been detected and would have led to a false laboratory report saying that the murder victim DNA had been found on an item collected in the suspect’s house. Defense lawyers need to think carefully about the potential for such errors because experience shows that they can and do occur.” (emphasis added)

Riley concurs, writing, “These controls don't offer protection against contamination occurring before the samples arrived at the PCR lab. Negative controls also can't rule out contamination of individual samples. The individual samples lack individual signs of contamination if it occurs.” The bottom line is that some instances of contamination probably go undetected.

Dishonesty involving controls

William C. Thompson delineates another problem:

“While most of the problems are due to inadvertent mistakes, a number of cases involving dishonesty have also come to light. DNA analysts have recently been fired for scientific misconduct, and specifically for falsification of test results, by a number of forensic laboratories, including labs operated by the FBI,¹⁴ Orchid-Cellmark (another large private DNA laboratory),¹⁵ the Office of the Chief Medical Examiner in New York City,¹⁶ and the United States Army.¹⁷ In all of these cases, the analysts were caught faking the results of control samples designed to detect instances in which cross-contamination of DNA samples has occurred.”

Footnotes for Thompson

14. See, U.S. Department of Justice, Office of the Inspector General, The FBI DNA Laboratory: A Review of Protocol and Practice Vulnerabilities, May 2004. Available online at: http://www.usdoj.gov/oig/special/0405/index.htm

15. Laura Cadiz, Md.-based DNA lab fires analyst over falsified tests, Baltimore Sun, Nov. 18, 2004.

16. Author’s interview with Robert Shaler, former Director of the OCME DNA Laboratory.

17. Associated Press, Worker in Army lab may have falsified DNA test result. Aug. 27, 2005; Memorandum For All Staff Judge Advocates Re: Brady Notice by Lisa Kreeger, Staff Attorney, Department of the Army, October 17, 2005 (on file with the author).

When the investigators and the DNA laboratory are in communication, the investigators sometimes make it clear that a certain result is desired. Thompson and colleagues (2003) write:

“Part of the problem is that forensic scientists refuse to take appropriate steps to ‘blind’ themselves to the government’s expected (or desired) outcome when interpreting test results. We often see indications, in the laboratory notes themselves, that the analysts are familiar with facts of their cases, including information that has nothing to do with genetic testing, and that they are acutely aware of which results will help or hurt the prosecution team. A DNA analyst in one case wrote:

‘Suspect-known crip gang member — keeps ‘skating’ on charges-never serves time. This robbery he gets hit in head with bar stool — left blood trail. [Detective] Miller wants to connect this guy to scene w/DNA …’

In another case, where the defense lawyer had suggested that another individual besides the defendant had been involved in the crime, and might have left DNA, the DNA laboratory notes include the notation: ‘Death penalty case. Need to eliminate [other individual] as a possible suspect.’”

The problem of having the laboratory be independent from the prosecution is a well-known one (http://learn.genetics.utah.edu/content/labs/gel/forensics/): “The [National Research Council] recommends that forensic DNA analysis be conducted by an unbiased outside laboratory that maintains a high level of quality control and a low error rate.”

Case Studies of Contamination from Co-Processing of Samples

Thompson gives two examples of suspect identification on the basis of dubious cold hits:

“In one particularly interesting Australian case, DNA on the clothing of a murdered toddler named Jaidyn Leskie was linked, via a ‘cold hit,’ to a young ‘mentally challenged’ woman who lived hundreds of miles away and who, by all accounts, had never left her own village. Police could find no way to link the young woman to the Leskie murder and at first dismissed the ‘cold hit’ as an ‘adventitious’ (coincidental) match. However, a coroner’s investigation established that DNA from the young woman had been processed through the same laboratory at about the same time as the toddler’s clothing. The young woman had allegedly been the victim of a sexual assault involving a condom. Although laboratory personnel maintain that accidental transfer of samples between cases is impossible, it now appears almost certain that the young woman’s DNA from the outside of the condom accidentally contaminated samples from the toddler’s clothing.

“The facts of some recent cases in the United States have also raised suspicions about false cold hits due to contamination across cases. For example, in 2002, while investigating the 1969 murder of University of Michigan law student Jane Mixer, the Michigan State Police Crime Laboratory in Lansing found DNA of two men on her clothing. The profiles were searched through a database and matched two Michigan men, Gary Leiterman and John Ruelas. Police immediately suspected that Leiterman and Ruelas had been involved in the murder, but there was a problem — Ruelas was only four years old when Mixer was killed and had been living with his parents in another city. According to news accounts, police could find no link between young Ruelas and Mixer.²⁹ That did not deter Washtenaw County Assistant Prosecutor Steven Hiller who charged Leiterman with the murder. Hiller ‘created a scenario placing a young Ruelas at the [murder] scene as a chronic nose-bleeder whose blood dropped on Mixer.’³⁰ There is, however, another possible explanation for this ‘cold hit.’ Examination of laboratory records revealed that known samples of DNA from both Leiterman and Ruelas were being processed in the Michigan State lab on the same day as the old samples from the Mixer murder.³¹ Both men were being tested in connection with other cases unrelated to the Mixer murder. Although the Michigan State laboratory maintains that cross-contamination of samples across cases was impossible, it seems a very strange and unlikely coincidence that two men who, according to the prosecutor, were present when Mixer was murdered in 1969 just happened to have their DNA tested (for other cases) on the very same day as samples from the Mixer case were tested. Leiterman was nevertheless convicted of Mixer’s murder in 2005.” (emphasis added)

Footnotes for Thompson

29. Maryanne George, Murder case mystery deepens. Detroit Free Press, Jan 15, 2005.

30. According to news accounts, Hiller offered no evidence to support this theory. Liz Cobbs, Judge raises possibility evidence may have been contaminated at State Police lab, Ann Arbor News, May 11, 2005.

31. Author’s interview with Professor Dan Krane (a defense expert in the case). Also, Thersa Mask, Mixer’s dad is clear on one thing, Detroit Free Press, July 13, 2005.

Contamination is surely an unlikely event, at least in that it happens less frequently than it does not happen. Yet, which is less likely in these two cases, contamination or the involvement of the suspect identified by these cold hits? And even if improbable, how does this speak to a reasonable doubt?

Borderline situations

Suppose that a technician runs evidentiary samples and reference samples in the same batch out of laziness or ignorance. Suppose a different technician runs these samples together knowing that there is a greater chance of contamination. These two cases are distinguishable only if one knows the intent of the technicians. The second case is close to outright evidence tampering, but the similarity of the two situations implies that there is no bright line between sloppiness and outright fraud.

Frequency of Contamination

This is difficult to estimate for a variety of reasons. Donald Riley reports that some laboratories omit controls or perform them in such a way as to lessen the chances of detection. Thompson reports that many laboratories fail to record instances of contamination in a log book, despite the advice of the FBI. Some labs take the overly sanguine approach that if they detect contamination, it is not really an error. Thompson writes, “The surprise for defense lawyers who have managed to gain access to these files is how voluminous they are. Errors occur regularly.”

Summary

• Forensic DNA contamination is a well-known phenomenon that good labs attempt to minimize, and to document when it does happen.
• PCR-amplified DNA is one of the most serious contamination threats in a DNA forensics lab.
• Keeping a clean DNA lab is a little like maintaining a sterile environment, only more difficult.
• It is difficult to estimate the frequency of contamination for a variety of reasons. However, many instances have been documented.
• Negative controls can be used to identify contamination, but they only detect contamination in blanks, not evidentiary samples.
• Fradulent DNA forensics often takes the form of falsifying negative controls.

Bibliography

John M. Butler, Forensic DNA Typing (2005), 2^nd ed., Elsevier.

Terri Sundquist and Joseph Bissetti

http://www.promega.com/profiles/802/ProfilesinDNA_802_11.pdf

Donald Riley

http://www.scientific.org/tutorials/articles/riley/riley.html

Nora Rudin and Keith Inman, An Introduction to Forensic DNA Analysis, 2^nd ed. (2002).

William C. Thompson 2006

http://www.nacdl.org/public.nsf/0/6285f6867724e1e685257124006f9177

William C. Thompson; Simon Ford; Travis Doom; Michael Raymer; Dan E. Krane (2003)

http://www.nacdl.org/public.nsf/698c98dd101a846085256eb400500c01/c1d63d2bd1a582cf85256e540074c146?OpenDocument&Highlight=0,rape

Dick Warrington Forensics April/May 2005

http://www.forensicmag.com/articles.asp?pid=41

Dick Warrington Forensics April/May 2009

http://www.forensicmag.com/articles.asp?pid=275