The likelihood of DNA contamination

Part 31 in the Knox/Sollecito case

The subject of DNA contamination has been a frequent topic of this blog. DNA contamination is again near the forefront of the Kercher murder and the second trial of Amanda Knox and Sollecito. The court-appointed, independent experts, Drs. Conti and Vecchiotti, have issued a report that raises the strong possibility of contamination. The odds that a forensic DNA sample are contaminated are hard to determine yet very important. Let us first examine a specific statement made with respect to environmental contamination and this case, then look at the question more generally. A subsequent entry will examine the collection practices of the Rome lab and Conti’s and Vecchiotti’s evaluation of their work in this case.

Professor Giuseppe Novelli, a researcher into medical genetics and forensic DNA profiling, said, «Il contaminante va dimostrato, dove nasce e dove è. Il gancetto contaminato dalla polvere? Più probabile che cada un meteorite e butti giù questo tribunale» "The contaminant needs to be demonstrated, where it comes from and where it is. The clasp contaminated by dust? It's more likely that a meteorite comes down and knocks down this courthouse." (translation by komponisto)

Let us assume that Dr. Novelli is specifically referring to household dust. The potential for dust being an issue in forensic DNA testing first came to light with the publication of a paper from Bonnie Brown and coworkers: Toothman et al., “Characterization of human DNA in environmental samples,” Forensic Science International 178 (2008) 7–15. These workers sampled dust from offices, research laboratories, and classrooms.

Figure 3 in this paper is an electropherogram of dust from a classroom, and it shows peaks of 1000 to 3500 RFU corresponding to 3-6 alleles in some loci associated with shorter pieces of DNA. As the length of the DNA fragments increases (moving from left to right on the elecropherogram), the height of the peaks decreased. The authors point out that this is consistent with DNA template that is partially degraded. The combination of a sample’s being a mixture and its yielding only partial profiles makes it very difficult to identify individual profiles.

The authors wrote, “Results of this study have implications regarding the processing of forensic samples. First, the presence of genotypeable human DNA in dust illustrates a significant
potential contamination source in forensic investigations. Twenty-five of 36 samples contained sufficient input human DNA for STR analysis using the AmpFlSTR® Profiler PlusTM assay (~1.0 ng), and 36% (including low-input samples) produced alleles at multiple loci. These results demonstrate that even though anti-contamination measures may be in place at a crime scene and the laboratory, trace DNA derived from dust in the vicinity of other evidence is capable of producing signals higher than background noise in STR analyses.”

Trace DNA and the environment
Secondary transfer is the movement of DNA from a donor to an intermediate object then to another object from which it is collected. Some forensic scientists only classify it as contamination when it occurs subsequently to the object’s being taken into custody; however, other workers prefer to treat innocent secondary transfer as equivalent to contamination on the basis that neither is relevant to the investigation. Both secondary transfer prior to an object’s being collected and contamination need to be understood more thoroughly for trace DNA to be used routinely in DNA forensics. Van Oorschot and colleagues wrote, “Greater effort needs to be made by police/crime investigators to investigate how a DNA sample arrived at the location where it was found, as well as by scientists to better understand the impact of activities on the relative amounts of DNA from particular sources at a crime scene… Some preliminary contributions to our knowledge of transfer in relation to residential burglary and street robbery have recently been made [67].

In their review of trace DNA in forensics Van Oorschot and colleagues suggested six remedies to address the problem of contamination:
“1. perform more studies similar to those of Raymond et al. [67], Cook and Dixon [202], Dowlman et al. [203] and Toothman et al. [201] in order to learn more about the occurrence and persistence of DNA on particular surfaces in different environmental conditions” Understanding how prevalent and persistent background DNA is in the environment is far from being completely understood. Dr. Novelli declined to comment for this report, but his comparison with a meteorite is premature, at best.

The relationship between DNA sample size and the ease of transfer
In their 2010 review article Van Oorschot and colleagues wrote, “Contaminant DNA may appear as either the major or minor sample within a mixture or, alternatively, may overwhelm the target DNA completely.” One explanation for this somewhat counterintuitive statement is that contamination in the laboratory may introduce DNA from previous PCR amplifications by a number of possible routes. However, the two main DNA profiles of interest (Meredith’s on the knife blade and Raffaele’s putative profile on the bra clasp) both involve relatively small amounts of DNA; therefore, we will focus on small samples.

The discussion of low copy number (LCN) testing from the Crown Prosecution Service noted, “This increased sensitivity means ultra-clean laboratories are needed for the testing to minimise contamination of the sample by DNA from any other source.” The New Zealand Institute of Environmental Science and Research has spent $1 million building anticontamination areas for low copy number (LCN) DNA forensics. The New Zealand Herald wrote, “The bogey is contamination. The very sensitivity of the technique which enables it to extract a DNA profile from the tiniest sample also makes it extremely vulnerable to contamination. Stringent measures are needed to minimise that risk… We live in a ‘soup’ of DNA, explains ESR forensic programme manager Keith Bedford. ‘If I were to shed dandruff, massive amounts of dna could fall ... hair could carry DNA. The way I am speaking at the moment, we could probably detect DNA on this pad in front of me.’”

Sara Gino testified for the defense in the trial of the first instance, and some of what she had to say is pertinent to this issue. From the Massei report (p. 258, English translation): “She reaffirmed that [the risk of] contamination exists, and emphasised that in minimal quantities of DNA there is not necessarily a greater risk of contamination but it was easier to notice the effects of the contamination and be misled (‘...It's not that the risk of contamination is greater; but it is easier to see the contamination...’ page 92).” In response to a question on this subject, Professor Dan Krane responded, “There is absolutely no question but that contamination is a much greater problem in LCN cases than conventional DNA testing. The reasons that it is a greater problem are both because it is easier to detect contaminants ([Sara] Gino's point) and because it is easier to transfer (and to transfer without knowing) smaller amounts of DNA than larger amounts of DNA.”

Some examples of DNA contamination
Farah Jama was a young man accused of rape on the basis of his DNA seemingly being found on the alleged victim. Mr. Jama is black and at 21 was too young to have entered the club at which the incident occurred, which catered to people over 28. Moreover, the alleged victim did not recall seeing a black man at the club that night. Yet, as Milanda Rout wrote, “But the judge and the jury did not buy his alibi, despite supporting evidence from his father, brother and friend. Instead, they believed the forensic scientist who testified there was a one in 800 billion chance that the DNA belonged to someone other than the accused man.” After Mr. Jama spent more that eighteen months in prison, he was released because prosecutors said that they could not rule out contamination. The contamination event may have occurred during two forensic medical examinations, one of the victim and the other of Mr. Jama on an unrelated matter that occurred one day earlier.

Perhaps the most thoroughly studied case of contamination is that which occurred in the Jaidyn Leskie case. This blog has covered the Leskie case on two previous occasions. The DNA of a woman who probably never left her village was found on the clothing of the submerged body of a toddler. She was a mentally challenged woman who may have been raped, which is why her DNA was being examined. Because the woman was such an exceedingly unlikely suspect, the only reasonable explanation was contamination. Contamination has been documented on several occasions when evidence items from unrelated cases are examined within a few days in the same lab.

Russell John Gesah was charged with the rape and murder of a mother and child. Kathleen Skeen wrote, “A Victorian Police Forensic Services Centre review found clothing with Mr Gesah's DNA from an unrelated offence had been examined on the same day and same surface as clothing from the Tapp case." The Gesah case, and the murders of Jane Mixer and Jane Durrua (see below) are all examples of DNA cold hits. The Gesah case prompted the State of Victoria to reexamine thousands of cases (see below).

Gregory Turner might have been convicted of murder on the basis of DNA evidence. However, a forensic worker contaminated a key piece of evidence with his and her DNA. She also acknowledged contamination in two other cases on which she had worked. An interesting aspect of the Turner case is that the DNA from the victim came from her fingernails, and Mr. Turner’s DNA came from his wedding ring. These facts suggest that the presence of liquids is not necessary to bring about cross-contamination, in contrast to the implications of Patrizia Stefanoni’s testimony in the present case.

The murder of Jane Mixer was initially attributed to a serial killer. When Gary Leiterman’s DNA was found on the decades-old evidence, he was convicted. However, the presence of the DNA matching then four-year old John Ruelas on the same item of evidence (despite Ruelas’s living in another city) strongly points to this being another example of contamination. This illustrates another important principle. One does not always know the precise moment that contamination occurred, but one can infer contamination when the direct deposit of DNA is shown to be highly unlikely.

A seemingly solved cold case that turned out to be contamination involved the 1968 murder of Jane Durrua. Jerry Lee Bellamy’s DNA was found when the evidence was tested in 1999. Evidence against Mr. Bellamy in an unrelated case was tested on the same day as items from the Durrua case. The actual evidence of contamination was not conclusive, but despite this, charges against Mr. Bellamy were dropped. Alleged serial killer Robert Zarinski was later arrested, but he died before he could be tried.

The difficulties in quantifying the frequency of DNA contamination
Not all labs document contamination events. Some labs argue that contamination that is identified with the use of negative control experiments does not count as contamination. Negative controls will spot wholesale contamination events but will not necessarily catch sporadic contamination. These facts make it difficult to quantify how frequently contamination occurs. However, it does not seem to be an especially rare event. Professor Thompson is a lawyer who specializes in probability theory as it relates to DNA profiling. Maura Dolan reported that he is among the leading authorities on laboratory errors in the United States. In response to a request from the Los Angeles Times to review the records from some California forensics labs, Thompson said, “’on a regular basis, laboratory personnel make mistakes that could lead to false identifications’ of suspects.” He also indicated that what has emerged in recent years is just “the tip of the iceberg.”

In 2008 Professor Thompson wrote an article, “The Potential for Error in Forensic DNA Testing (and How That Complicates the Use of DNA Databases for Criminal Identification"). “Doubt was also cast on a number of convictions in Queensland when a forensic scientist who had previously worked for a state forensic laboratory publicly expressed concerns about the reliability of the lab’s work. He told The Australian newspaper that it was not uncommon for the lab to mix up DNA samples from different cases.[62] For example, he said that analysts’ own DNA, from blood samples used as analytical controls, often was mixed up with (or found its way into) casework samples, creating false matches: “[Q]uite often my (colleague) would walk down the aisle and say, ‘I’ve just committed another rape on the Gold Coast.’”[62] The analyst said that while many such errors were caught, sample limitations made it impossible to resample or retest in some questionable cases.” These remarks underscore the notion that DNA contaminations are not a rare event.
[62. A. McDonald, “DNA evidence claim clouds Australian convictions,” The Australian, July 8, 2006.]

In response to the Russell John Gesah contamination incident (see above), the Victorian police reexamined their cases involving DNA forensics. During the period from 1988 to 2008 the Victorian police service handled 7000 cases involving DNA. According to Peter Gregory and coauthors, “In 2003, Mr Scheffer told an inquest on Moe toddler Jaidyn Leskie that since late 1999, 39 cases had been identified as requiring "diagnostic and corrective action", with most involving contamination.

Finally, testimony reported by Annabelle McDonald (in The Australian) implied that mixing up samples is a not uncommon event. Although mislabeling of samples (if that is what mixing up means) is not itself contamination, it has the potential to lead to the same erroneous judicial result. A mislabeling in Nevada was uncovered during an independent review of the Lazaro Sotolusson case. In addition Dwayne Jackson was also the victim of a similar mistake at the Las Vegas forensics lab.

Conclusions
There is not yet enough information on environmental contamination to make conclusive statements about how common environmental contamination is; however, DNA is deposited routinely in all sorts of ways that are unrelated to criminal activity. The authors of a recent study believe that environmental dust can give rise to extra alleles in evidence samples. The frequency of contamination is difficult to quantify, but it is not an especially rare occurrence. The chances of contamination are greater for DNA in the low template range than they are for larger samples. Historical examples of contamination suggest that it is more likely to occur when items of evidence are processed closely in time. Contrary to the implication of Dr. Novelli's remarks, it is rarely the case that the exact mechanism of contamination is proven.

Things said and left unsaid: Barbie Nadeau’s coverage of the Knox/Sollecito case (Part XIII)

Ms. Barbie Nadeau’s book on the murder of Meredith Kercher is now out and will be reviewed here soon. A reading of some of her Newsweek and Daily Beast articles may give a glimpse of what to expect. Early in the article Ms. Nadeau wrote, “Just days after Kercher's body was found last November in the villa Knox shared with the victim, security camera footage showed the couple buying lingerie in a local store, with Knox giggling and telling Sollecito, ‘Afterwards I'm going to take you home so we can have wild sex together.’” Ms. Nadeau’s account is misleading in several respects. Ms. Knox did not have access to her own belongings at this time (her flat was a crime scene) and needed to buy underwear and borrow jeans. The security camera did not have sound, and witnesses say that the store has very loud music, making it difficult to overhear someone’s conversation. Whether the merchant, who was a paid interviewee and the actual source of the quote, spoke English or not is disputed. However, the couple did kiss in the store, and their demeanor appears playful.

Ms. Nadeau went on, “And by her own account in a prison diary leaked to the media, she details her sexual escapades with at least seven men she'd been with in her three months in Italy before her arrest. She even wrote that she might have HIV and then she uses a process of elimination to narrow down who might have given it to her. The diary is part of the collection of evidence and could be damaging to Knox in a court trial. Recently leaked segments of her continuing tome paint a disturbing picture of her state of mind. In one entry, she writes: ‘I think it's possible that Raffaelle went to Meredith's house, raped her, then killed her and then when he got home, while I was sleeping, he pressed my fingerprints n the knife.’”

Mark Waterbury rightly took Ms. Nadeau to task for the many problems with her assertions. Ms. Knox wrote of having seven intimate partners her whole life because she was told that she was or might be HIV-positive. Ms. Knox’s diary shows her emotional distress, and to claim that it “details her sexual escapades” is so blatantly wrong that it leaves one wondering what Ms. Nadeau could possibly have been thinking. Some of the most problematic assertions, including the incorrect description of the night of the murder, were based on an English translation of an Italian translation of her diary, instead of the original. When she was contacted about the mistranslation, Ms. Nadeau claimed that there was a second diary, but she is the only reporter who has made this claim to the best of my knowledge.

The last portion of the article says as much about Ms. Nadeau as about the case. “To complicate things further, Sollecito and Knox's reported break up prompted speculation that they would testify against each other. But Sollecito's flowers may be a signal that their defense teams may actually work together to pin the blame on Guede. If that strategy succeeds, the cipher of Amanda Knox may never truly be decoded.” Ms. Nadeau’s use of the word “pin” suggests that she believed Knox and Sollecito were guilty. Her linking the potential success of their blaming Mr. Guede to the potential failure to decipher Ms. Knox leaves little doubt about her opinion on their culpability. Why Newsweek entrusted their coverage of this case to her is the real mystery.

Let us consider some of her entries at The Daily Beast more briefly and with comments interspersed. In a recent article Nadeau wrote, “Shortly after her arrest, Knox admitted to being at the Perugian villa when Kercher was murdered. The confession was later thrown out of the body of evidence because of allegations of police brutality and coercion, but much of what she said in that original statement coincides with known elements of the case, including evidence gathered by CCTV footage from a nearby parking garage.” The CCTV footage may be of Meredith entering her cottage for the last time. Yet it is what this article does not say that is at least equally troubling. Nothing about the police giving out false information and only a little to on the British press, which sometimes behaved outrageously. Meredith’s father, John Kercher, was part of the British tabloid press corps, and this may explain some of the contempt the British press exhibited toward Ms. Knox.

Shortly after the conviction, Ms. Nadeau began an article, “Is the real Amanda Knox the sex-obsessed, cold-blooded murderer that the prosecution depicted? Or worse?” This question and the title of her article are truly egregious, worthy of comparison to the Newsweek mug shot cover on the Duke lacrosse case in 2006, and that is saying something. Clearly Ms. Nadeau had access to Edda Mellas and Curt Knox, which undercuts her claim about being shut out in the article about how the media got the case wrong (see above). The rest of the article lacks a critical appraisal of the forensics. “They [the jury] wondered why Amanda’s DNA and Meredith’s blood was wiped away and recovered with Luminol. They thought it was Raffaele’s bloody footprint on the bathroom rug and his DNA on Meredith’s bra clasp. They believed the prosecutor’s testimony that Meredith’s DNA was on the blade of the knife that had Amanda’s DNA on the handle.” There is no reason to assume that a substance that tests positive with Luminol is blood; it is a presumptive test that needs to be confirmed with one that is more specific for blood.

Immediately after the conviction Ms. Nadeau wrote, “At one point the stepmother of Raffaele Sollecito, Knox’s former boyfriend who was sentenced to 25 years for his part in the murder, yelled out ‘F@#k you,’” Ms. Nadeau is the only reporter who heard this; others heard, “Forte, Raffaele,” which just means strength, Raffaele. “But in the end, the two judges and six lay jurors trusted the sanctity of the DNA evidence, and doubted the words of Knox and Sollecito….For those who believed that Knox was guilty, the most damning evidence was the presence of her DNA mixed with Kercher’s blood in the house, and her statement that she was in the house at the time of the murder.” Ms. Nadeau has never written about the open letter signed by nine DNA forensics experts, but even if she is unaware of their views, she should know that the quality of the DNA evidence is very much in dispute.

Ms. Nadeau wrote about the jury’s report on its reasons for conviction, “A spot of Knox and Kercher’s mixed blood in one of the bedrooms, found using Luminol, and four additional spots in the small bathroom the girls shared also swayed the jurors.” The claim that finding Knox’s DNA mixed with Kercher’s blood means that their blood was mixed is dubious.

Quoting prosecutor Mignini Nadeau wrote, “’The window was broken from the inside, not the outside. The glass was on top of the clothes that had been strewn around the room, not under them. The break-in was staged and Knox is the one who did it.’ He also hinted that Knox and Sollecito might have been in a drug-fueled frenzy when they allegedly killed Kercher. He outlined the effects of cocaine and acid, and told the judges and jury how Knox and Sollecito ran with a crowd that often used these ‘stupificante,’ or stupefying drugs.” What drugs? Knox and Sollecito acknowledge smoking marijuana but no evidence of other drugs was presented. The notion that the window was broken from the inside is not convincing. Ms. Nadeau presents this closing argument without commenting on its many problems. This is only a reasonable approach if we accept this blog entry as pure reporting (with Ms. Nadeau reporting and editorializing are sometimes mixed. And yet after the introductory paragraph, eight of the nine succeeding paragraphs cover Mr. Mignini’s summation, whereas only one covers the defense’s summation.

“Other evidentiary problems include the lack of video or audio tapes of Knox’s original interrogation and the scientific police forgetting to pick up a bra clasp for nearly six weeks. That clasp is said to have Sollecito’s DNA on the metal hook and is the only material evidence police have tying Sollecito to the crime. While these obvious errors might well establish reasonable doubt in an American courtroom, it is unlikely they will have the same effect here in Italy, where defendants often seem to be presumed guilty until proven innocent.”

“Last July, testimony by noted forensic specialist Adriano Tagliabracci was abruptly suspended after the defense discovered that they had not been given all the prosecution’s evidence reports. Specifically, they did not have crucial evidence about Sollecito’s DNA found on the clasp of the bloodied bra that was cut from Kercher after she was stabbed.”

“Saturday’s hearing ended with a dramatic exchange about whether the prosecution had given the defense key documents regarding the DNA on Kercher’s bra. The charge was seen as a blatant attempt by the prosecution to throw the defense’s witness, and it worked. The exchange ended with Sollecito’s lawyers accusing the prosecution with illegality—a move many thought was primarily to set the stage for an appeal if one or both are convicted.” These three examples show that Ms. Nadeau does report on some aspects of the case that create sympathy for the defendants.

Ms. Nadeau wrote an article about Rudy Guede, who was the only person at that time convicted of Meredith Kercher’s murder. “’He has never changed his story,’ Guede’s lawyer, Valter Biscotti, told The Daily Beast this week. Biscotti would not supply any details about the version of events Guede gave at his own, closed-door trial, but he did say, ‘He has always maintained that they were all there, but that he is not the one who killed her.’” Ms. Nadeau did not comment on this remarkable assertion. When he was in Germany, the police intercepted one of Mr. Guede’s skype conversations in which he said that Amanda was not there. The first months after his arrest he said that an unidentified man murdered Ms. Kercher when Guede was on the toilet. Months later he began to claim that the man was Raffaele Sollecito and that Ms. Knox was at least present. ABC news reported that “Guede, 25, has at different times said the Knox, 22, of Seattle, was and was not at the house the night of the murder. At one point, he also implied that Knox's co-defendant Raffaele Sollecito was at the murder scene.”

Finally, I will expand upon a point I raised in the comments section of the previous article. In response to a question during a live chat, Ms. Nadeau said, “I think it is important to remember that the same scientific police and laboratories tested the DNA for all three suspects. That is to say, if the DNA matches Rudy and is accepted, then the DNA that matches the other two should also be accepted. How she may have left so little DNA if she was actually in the room is very hard to square.”

Ms. Nadeau is asserting implicitly that all DNA evidence is equal, but this is false. We know that the knife profile is weak, has drop-ins, and has considerable imbalance in intensity between most of the pairs of alleles. We know that the bra clasp was left in an insecure location for 47 days and handled poorly when it was finally collected. As Dan Krane said, “The science of DNA profiling is sound. But, not all of DNA profiling is science.” The evidence against Guede has never been released, and one cannot pass judgment on its quality without seeing it. There is second problem with Ms. Nadeau’s assertion. Rudy Guede was only identified as a suspect after the forensics came back, but Raffaele Sollecito and Amanda Knox were already suspects (first on the basis of demeanor, then on the basis of their interrogations) before the forensics was completed. This problem is particularly acute for the bra clasp, and one has to consider the problem of confirmation bias or even outright fraud when weighing the forensic evidence. The third problem is that even within a lab, the quality of work may not be uniform. For one thing lab contamination can be a sporadic event, as it was in the Jaidyn Leskie case. For another, there may be a lone sloppy or dishonest technician in a lab full of competent, ethical ones.

Update 20 April 2010
I added a hyperlink to the Newsweek story in paragraph 1 and the word "to" in paragraph 6, line 4.