Secondary transfer of DNA and DNA contamination

Part XV in the Knox/Sollecito case

Introduction
This post returns to the subject of forensic DNA contamination. It will define primary and secondary transfer of DNA and explain how they are related to contamination. It also treats clerical errors and the need for independent review of the forensic DNA data. It reconsiders the lessons of the Leskie case. These concepts will be applied to some of the DNA forensics in the murder of Meredith Kercher, especially as it relates to the evidence against Amanda Knox and Raffaele Sollecito.

DNA contamination
People sometimes confuse DNA contamination with secondary transfer of DNA. Let us define these terms and give a few examples. In Forensic DNA Typing, p. 152, John Butler wrote, “Contamination implies the accidental transfer of DNA.” Implicit in his definition is that the transfer happened after the evidence was collected. 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.”

Primary transfer of DNA
According to forensic scientist Suzanna Ryan, “Primary transfer is described as the transfer of DNA from an individual to an item.” Ryan summarized some of the factors that contribute to how much DNA is transferred: “Through studies by Ladd, et al and others, many in the forensic community believe that some individuals just naturally shed more cells when handling an item (‘good’ shedders) than others (‘poor’ shedders). Other factors including the substrate being handled (rougher items collect more DNA), the time since the individual last washed their hands, how nervous the person is (nervousness can lead to increased sweating), and how often they touch their mouth, eyes, hair, face or other body parts (thus gathering DNA on their hands) play a heavy role in whether a DNA profile will be obtained through simply touching an item.” One presumes that rougher items cause more cells to transfer than smoother items, but Ms. Ryan did not give specific examples.

Secondary transfer of DNA
Suzanna Ryan wrote, “In secondary transfer, there is no direct contact between a person and an object. It is instead transferred through an intermediary. This could be another person or another object. An example—expanding upon the same knife example as above—is as follows: Person A shakes Person B's hand. Person B touches the handle of a knife. Secondary transfer theory would allow that Person A's DNA could be transferred first to Person B's hand and second to the knife handle. This would mean that even though Person A never actually touched the knife handle, his DNA could be present on it.”

Secondary transfer at the scene and the lab
Suzanna Ryan continued, “At the laboratory, the analysts are careful to wear gloves when handling evidence items. Great care is generally taken to clean scissors, tweezers, and other utensils between testing items. But what about items that are not cleaned as regularly? For example, is it possible to transfer DNA from an item of evidence to a ruler, and then when the next item of evidence is examined and photographed, could the DNA transfer again from the ruler to this item? A 2006 study by Poy and van Oorschot showed an example of secondary transfer when a mixed DNA profile was found on a swab taken from an examination magnifying lamp. This profile was searched in the lab's database and a match was found with a case that had been worked on the bench-top with the magnifying lamp. It was determined that DNA was transferred from the item being examined to the analyst's gloves and then onto the top of the magnifying lamp.”

This incident should be considered both secondary transfer of DNA (the item transferred DNA to the glove, which transferred it to the lamp) and DNA contamination because it happened in the analysis lab. This incident also illustrates how easy accidental DNA transfer can be in a lab. DNA contamination occurs when an analyst transferred his or her own DNA onto an object, but this would be classed as primary transfer.

Sometimes DNA contamination occurs in ways that do not involve the item of evidence. DNA profiles have been observed because an item of labware (plastic tubes, cotton buds, etc.) was contaminated with DNA from a person who helped to manufacture the item. The chemical reagents and equipment needed to perform the polymerase chain reaction, the DNA amplification step, are occasionally contaminated. These are examples of contamination that are due neither to primary nor to secondary transfer.

Primary and secondary transfer when several people handle an object
In the journal Nature, 387, p. 767 (1997), R. van Oorschot and M. Jones wrote, "Objects handled by many individuals all produced profiles with multiple alleles of varying intensity. To determine the effect of multiple handlers, we exchanged polypropylene tubes between individuals (2 or 3, 10 min each) with different genotypes. Although the material left by the last holder was usually present on the tube, that of previous holders was also retrieved to varying extents. The strongest profile obtained was not always that of the person who last held the object, but was dependent on the individual. We regularly observed profiles of previous holders of a tube from swabs of hands involved in these exchanges, showing that in some cases material from which DNA can be retrieved is transferred from object to hand (secondary transfer).” This article was the first one of which I am aware to document that DNA profiles could be recovered from fingerprints. This may also be the first one to use the term “secondary transfer.”

Primary and secondary transfer of DNA to the bra clasp
Some have argued that the amount of Raffaele Sollecito’s DNA on the clasp can only have been deposited through primary (direct transfer), not contamination or secondary transfer. This argument is false on several grounds. It ignores that that the amount of DNA in primary transfer depends upon several factors (see above). It ignores that contamination can produce strong profiles that analysts have mistaken for genuine ones (see below). It ignores the fact that Mr. Sollecito’s DNA profile on the clasp was relatively weak in intensity, roughly 200 RFUs. Perhaps most fundamentally, this argument runs counter to a principle of DNA forensics. As part of his analysis of the Leskie case (see below), forensic scientist Dan Krane wrote, “In fact, it is quite uncommon for DNA tests themselves to say anything about the circumstances (or even the time frame) associated with the transfer.” Jason Gilder wrote in an email to me, “One of the standard axioms of DNA typing is, the presence of a DNA profile says nothing about the time frame or the circumstances under which DNA was transferred to that item. Contamination falls into is one such instance of an issue that cannot be identified by the electropherogram alone (unless you are dealing with a control sample or other known profile).”

On the other hand, an anonymous commenter on a discussion board offered this hypothetical scenario for secondary transfer of Mr. Sollecito’s DNA: “For example, let's say that DNA from Meredith, Amanda and Raffaele was on the hand towels Rudy took from the bathroom (highly probable, since they'd all used the bathroom in the days before the murder). Drying your hands on a hand towel is exactly the kind of rubbing action that would shed DNA. If Rudy had trodden on one of the towels, then trodden on the bra clasp (one of the hooks was deformed, of course) that could have transferred DNA from the towels to the hook, with no direct contact from Raffaele necessary. In turn, Raffaele's DNA was more easily identified because they could use the more sensitive tests for the Y chromosome, whereas the other DNA would have been mixed female and thus tricky to correctly identify.” This is a speculative scenario, but it is not the only kind of secondary transfer that one could imagine. Sollecito’s DNA was likely on the door to Meredith’s bedroom, for example, because he tried to break the door down.

Clerical errors in DNA forensics
Previously we have explored why the electronic files are important. However, simply having an independent expert review the case file can catch some types of errors even without the electronic files. DNA forensic consultant Norah Rudin encountered a case where two reference samples were switched, due to a clerical error. Lazaro Sotolusson was held for a year and would have been tried for rape involving juveniles, facing multiple life sentences. In a preliminary hearing, one of the female victims identified Sotolusson as her attacker, possibly a case of a mistaken eyewitness. Dr. Rudin examined handwritten notes and computer files (not the sort of files one uses to create a DNA electropherogram, such as a .fsa file) and concluded that the lab had switched the two samples. The lab retested samples from Mr. Sotolusson and Joseph Coppola and confirmed the error. Rudin stated to a reporter, “If I hadn't looked at the original data, I likely wouldn't have found the mistake. That's one of the reasons that I always look at the original data… I believe every case needs to be reviewed by an independent analyst.” (emphasis added)

Errors of a similar kind have occurred elsewhere. Professor William Thompson wrote, “I recently reviewed the corrective action file for an accredited California laboratory operated by the District Attorney’s Office of Kern County (Bakersfield). Although this is a relatively small laboratory that processes a low volume of samples (probably fewer than 1,000 per year), during an 18-month period, it documented multiple instances in which (blank) control samples were positive for DNA, an instance in which a mother’s reference sample was contaminated with DNA from her child, several instances in which samples were accidentally switched or mislabeled, an instance in which an analyst’s DNA contaminated samples, an instance in which DNA extracted from two different samples was accidentally combined into the same tube, falsely creating a mixed sample, and an instance in which a suspect tested twice did not match himself (probably due to another sample-labeling error).” (emphasis added)

Whether or not an independent analysis would have caught possible clerical errors here is an open question. The Knox defense team asked for an independent analysis of all of the forensic data, but the judge denied the request. As long as human beings are part of the testing process, clerical errors are likely to happen at some frequency. This factor and the possibility of contamination ought to be considered when one ponders the astronomical odds that are sometimes used when describing a particular DNA result.

The Leskie case and its relationship to the knife and bra clasp as evidence
Jaidyn Leskie was a toddler who was murdered in Australia over a decade ago. A DNA profile belonging to a Ms. P. was found on two pieces of Jaidyn’s clothing, a bib and some track pants. Ms. P’s profile had varying intensities at different loci, suggesting sample degradation. However, her profile was quite strong in some loci, having peak intensities of over 1000 RFUs. Another example of contamination occurred in State of Illinois v. Sean Evans (2003). In this example a positive control was contaminated with a sample containing a DNA ladder, a series of DNA fragments of increasing length that might be used as a calibration standard. Note that the intensity of the fragments making up the ladder is 500-1500 RFU, similar to a typical evidence sample. The unusual nature of the ladder DNA helps to show that contamination occurred, but this is seldom true for other kinds of contamination.

Ms P’s DNA profile was ultimately attributed to contamination partially on the basis that Ms. P lived hundreds of miles away from the crime and never left her village. Yet she could have lived next door to the Leskie family, and contamination would still be a likely explanation for finding her DNA on the toddler’s clothing. Jaidyn’s body was found submerged in water for months. These conditions are antithetical for the preservation of DNA evidence. Significantly, none of Jaidyn’s DNA was recovered from his clothing. This should have been a reason to suspect contamination on its own.

The bloodless kitchen knife in the Knox/Sollecito case is similar to Jaidyn’s clothing in that no human DNA was likely to have been on the blade at the time of collection at any section that tested negative for blood; therefore, the DNA was most likely deposited in the lab. The Leskie case was one in which the electronic data files were particularly helpful. The Knox/Sollecito case would also benefit by independent examination of these files. For example, one could use the .fsa files to examine other samples along the knife blade and handle using the same threshold value for all samples. One might find profiles belonging to other individuals, and this would strengthen the argument that DNA on the knife arose from contamination or secondary transfer.

Could contamination have occurred in the Leskie case? The evidence with Ms P’s DNA was examined within a few days the evidence from the Leskie case, and Dr. Krane noted “even if evidence samples from the two cases did not come into direct physical contact, secondary transfer (by way of common contact with a surface or implement) of biological material from the condom or articles of Ms. P’s clothing in the rape investigation to the bib and track pants could have occurred.” The lab in question had argued that contamination was unlikely on at least two grounds, one of which was the lack of DNA from Ms P showing up in other profiles. Dr. Krane agreed that “no other samples including negative controls associated with both investigations display indications of Ms. P’s allele’s being present.” However, he rebutted the lab’s point by stating, “there is no good reason to expect that contamination would uniformly affect all samples if and when it does occur. In much the same way, Salmonella-tainted meat placed on one part of a countertop at the beginning of food preparation does not mean that all food subsequently prepared for the same (or even a later) meal will be similarly tainted – just those items that also come in contact with the same portion of the countertop can be reasonably expected to have a chance of being tainted.” Although the amplification of DNA through use of the polymerase chain reaction (PCR) creates the possibility of contamination via the amplified DNA, Dr. Krane did not think it is as likely in this case as secondary transfer.

There is one more point with respect to the bra clasp that needs to be addressed. It is sometimes argued that Mr. Sollecito’s DNA could not have arrived on the clasp by contamination because his DNA was found on no other items except for a cigarette butt. This is essentially the reasoning of Judge Micheli, who oversaw Rudy Guede’s trial. From the Wikipedia article on the murder of Meredith Kercher, “The judge at the trial of Guede acknowledged that the DNA sample was considered small, but described the claim of contamination at the laboratory as making ‘no sense’, since there was no material from which such contamination could have come, and so ‘the risk would have been the loss of traces found there, not the risk of somehow discovering new traces.’” This argument is false for at least two reasons. It ignores the possibility of contamination from Mr. Sollecito’s DNA in the lab, and it ignores the empirical facts of the Leskie case, where Ms. P’s DNA did not show up in other items or controls.

Summary
One way to distinguish between secondary transfer and contamination is to say that the difference between primary and secondary transfer depends upon how the DNA was deposited on the evidence item, but contamination depends upon when the DNA was deposited: if the DNA were deposited before the item was collected as evidence, it would not be contamination. We have also seen that contaminating DNA does not evince a difference in intensity that would allow it to be distinguished from DNA arising from primary or secondary transfer before the item was collected as evidence. Clerical mix-ups are another source of error in DNA forensics, and catching them is one of many reasons to welcome independent reviews of the results. Raffaele Sollecito’s DNA could have arisen on the bra clasp from contamination or secondary transfer, as well as primary transfer. Meredith Kercher’s weak, dubious DNA profile on the knife is almost certain to have arisen from contamination.

Updates 1/13/2012 and 1/19/02

I corrected Suzanna Ryan's name from Suzanne, and I fixed a link to one of her articles. I added a quotation mark and fixed a link to William C. Thompson's article.

The prosecution's failure to release the electronic data files to the defense

Part XIV in the Knox/Sollecito case

(updated three times on 4/27/10, once on 5/6/10, and once on 5/20/10)

One of the first things that drew me to the Knox/Sollecito case was the open letter on two items of evidence, the kitchen knife and the bra clasp. The two coauthors, Drs. Elizabeth Johnson and Gregory Hampikian are forensic DNA scientists. A one-paragraph appendix to the open letter was co-signed by seven additional experts in the forensic DNA field, including Dan Krane and Jason Gilder. The open letter completely dismissed the DNA evidence linking the knife to the crime and said. “Handling and movement of this sample [the clasp] has compromised its probative value.”

Dr. Johnson was initially approached by someone close to the defense team about looking into this case, but I have no information on how the other individuals became involved. The defense gave Drs. Johnson and Hampikian the limited materials that were released in discovery; however, the other seven did not have these data, owing to time. The seven co-signers wrote, “…While I have not had the opportunity to review all of the case-specific data in this matter, I agree that the scientific evidence that they describe does not convincingly associate the kitchen knife with the murder of Meredith Kercher. I also agree that the handling and movement of the bra clasp that they describe would have compromised its probative value, and that the presence of a DNA profile does not speak in any way to the time frame or the circumstances under which DNA became associated with this sample…” The seven names appear at this website but not on the pdf file of the letter itself; however, several of them have confirmed their involvement in the case in email correspondence.

One portion of the open letter made specific mention of the lack of release of the electronic data files. Drs. Johnson and Hampikian both requested the release of the electronic files used to construct the DNA electropherograms (for one manufacturer, these are called .fsa files), according to Jason Gilder. Dr. Krane did so on multiple occasions, according to Jason Gilder, who is one of the seven co-signers of the open letter. Dr. Krane had previously put electronic data files to very good use in the Leskie case; therefore, it is no surprise that he would request them here. The requests were made via the defense lawyers, but the prosecution steadfastly refused the requests. According to Dr. Krane, the release of electronic files is the almost universal norm. Dr. Norah Rudin provides on her website a standard form, DNA discovery request, for cases involving DNA forensics. From this document:
“2. Items #7 and #8 listed in the Discovery Request refer to electronic data, also known as raw data.
a. Electronic data is considered standard discovery and is critical to an independent review.
b. Electronic data must be received prior to commencing an independent review.
c. The laboratory will typically provide you with a CD containing electronic data.”

According to Amanda’s stepfather Chris Mellas, the defense team had asked for the DNA forensic data (this would be expected to include the electronic files and machine logs) but was told that they did not exist. Then the defense learned that the Kercher family’s lawyer had some of this information and demanded that the judge order the release of the data in the summer of 2009. The prosecution released some data, but not what was asked for. The lack of release of these data was one of the reasons for a mistrial motion in the fall of 2009. In April of 2010 Bob Graham wrote, “It has also emerged that the prosecution has failed to deliver to the defence all the paperwork and documentation related to the forensic testing. Chris Mellas, Knox's step-father who is currently in Perugia, said yesterday: ‘Our lawyers asked for everything, every file and record relating to the forensic testing. We were given some of the stuff, like what was on Meredith's shoes or a juice glass but not the full reports on the knife used or the bra-clasp.’ Deputy prosecutor Manuela Comodi brushed off the request for all forensic documentation and added: ‘They have everything they need. That is enough.’” Ms. Comodi’s words imply that the defense did not receive everything, just what the prosecution claims is enough.

The news reports from last summer and fall are consistent with Mellas’s recounting of the events. The defense did not know until last July that Patrizia Stefanoni had written the words, “Too low” on files relating to the DNA profile culled from the knife. ABC news reported in September of 2009, “The trial reopened with an attempt to have the case thrown out, but it was rejected after the judge and the jurors deliberated for 90 minutes. Lawyers for Knox and Sollecito became animated in their assertion that evidence had been withheld from them. Sollecito's lawyer, Giulia Bongiorno, addressed the court for 20 minutes arguing that the defense was not provided with crucial details of Sollecito's DNA allegedly found on Kercher's bra hook until July 30, 2009. The rights of the defense were damaged, she said, when ‘documents regarding the quantity of biological material on the bra hook and documents regarding the procedure used to attain DNA results were not made available to the defense.’”

Sara Gino, a defense expert witness, still did not have the dates on which the samples were run as of September of 2009. Frank Sfarzo reported in October of 2009 that the defense also asked the judge for an independent review of the forensics, which was not granted. Bob Graham reported in December of 2009, “Other forensic experts in several countries – including two from Britain – have started to study the DNA results but have delayed giving a verdict until they have received precise details on the methodology used by their Italian counterparts. The failure of the prosecution to provide these details to Knox and Sollecito’s defence teams is one of their central complaints.”

Do the defense team and the experts it consults have a right to obtain the data in electronic form? Release is clearly routine in the United States, consistent with the principle of transparency, enunciated by Keith Inman and Norah Rudin. Good science does not change when one crosses the border between nations. As an anonymous DNA forensic expert speaking about paper copies of the DNA forensic data said, “Certain parameters have irrevocably been applied to the data; as such it represents an interpretation by the laboratory rather than the original data.” A defendant should have the right to see and to challenge the evidence against him or her, and that principle is at risk in the Knox/Sollecito case.

Update I, 4/27/10: I corrected a minor typographical error in the first paragraph, where Dr. Johnson's name appeared twice.

Update II, 4/27/10: According to Chris Mellas by the time the defense learned that Mr. Maresca, the attorney representing the Kerchers, had some of the DNA forensic data, they had already requested it twice, before the pretrial and before the trial itself. Their third request in the summer of 2009 resulted in a court order that was not followed. To the best of my knowledge the additional DNA experts associated with the open letter made their requests after this time.

Update III, 4/27/10: Joy Halverson and Marc Taylor have confirmed that they signed the appendix to the open letter. Marc Taylor wrote, “We have discovered numerous manipulations of the data analysis or the actual physical analysis of the evidence by reviewing the electronic data…The electronic data is clearly the ‘best evidence’ in the legal system.”

Update IV, 5/6/10: In response to my query to another person who signed the letter, Simon Ford confirmed that he signed the letter and replied, “In my opinion, it is always important to review the electronic data underlying a test result, if only to confirm the integrity of the data set. In low level DNA cases such as this, in which the slightest contamination could compromise the test result, it is absolutely essential to review the electronic data for the key samples and the associated controls to check for low level contamination. I cannot think of any valid reason why the prosecutor would chose not to provide the electronic data. In my experience, forensic DNA analysts in the US are invariably happy to turn over the data to support their findings.”

Update V, 5/20/10
The Irish Times - Saturday, February 27, 2010
DNA testing project offers fresh hope to the wrongfully convicted

A forensic genetics expert, Dr Hampikian’s expertise is called on by the Innocence Project all over the US and further afield. He is currently working with the family of Amanda Knox, the American student jailed in Italy last year for the murder of British student Meredith Kercher.

A professor of genetics at the University of Boise, Idaho, Dr Hampikian’s role with the Innocence Project is voluntary, but one he takes seriously. He has been involved in four exonerations over the past decade, though, ironically, his DNA testing has confirmed guilt in two further cases.
“I can’t tell who is telling the truth or who is lying, but the DNA can tell,” he says. “Sometimes the Innocence Project is a bit of a misnomer. In two of the four exonerations, I developed evidence that led to the arrest of somebody new for the crime, so for some people we are the Guilty Project – and that’s an important part of what we do.”

This documents that Dr. Hampkian, like Dr. Johnson, is a consultant for the Knox defense team.

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.

The utility of electronic data files in DNA forensics cases

Part XII on the Knox/Sollecito case

“Full disclosure allows a thorough independent review of results and thereby helps assure that the underlying scientific methods are strong and appropriate, and that the laboratory’s interpretations are fair and accurate. Until the electronic data are made available, I cannot say that I have performed a complete review of the underlying evidence in this matter.”
-William Thompson, Victoria State Coroner’s Inquest into Death of Jaidyn Leskie

In a previous post (http://viewfromwilmington.blogspot.com/2010/03/interim-evaluation-of-evidence-against.html) I wrote:

Why are the .fsa files important? Technical bulletin 40-035 from Chromosomal Laboratories, Inc., is a checklist of what the laboratory is expected to provide, and it states, “Copies of all data files used and created in the course of performing tests and analyzing data in this case, including .fsa files, if applicable. These files should include all data necessary to independently reanalyze the raw data.” In response to a question on this subject, Professor Dan Krane wrote, “The biggest concern that I personally have regarding this case is the refusal of the prosecution to provide the defense with a copy of the electronic data that underlies the DNA test results -- that is virtually unheard of world-wide today and it would be especially important to review that data in a case such as this which seems to involve such low level samples.” According to a knowledgeable source, “Each tested sample has its own file. The file contains the full electropherogram trace information along with other information about the testing conditions (e.g., date, time, injection time, voltage, temperature, current, the RFU threshold used by the analyst). If you have the electronic data, you can use the DNA analysis software (GeneScan & Genotyper or GeneMapper ID) to independently analyze the electronic data. That allows you to examine the results as closely as possible (zoom in on the electropherogram to evaluate low-level results) and establish the RFU threshold of your choosing.” (emphasis added)


The knowledgable source I quoted above is Jason Gilder, and he and Dan Krane are cosigners of the open letter from a total of nine forensics scientists that is critical of the evidence in this case. I would like to reexamine this issue with respect to how these files are useful. I will have more to say about circumstances of their being withheld from the defense in the future.

Contents of fsa files
The fsa files are created by the DNA analysis instruments of Applied Biosystems, and the program GeneScan can read them. These files contain the times of and dates on which samples were (runhttp://www.nfstc.org/pdi/Subject06/pdi_s06_m01_02.htm). They also contain the peak detection threshold in relative fluorescence units. Perhaps most importantly, they contain the electronic information needed to recreate a DNA electropherogram with the parameters chosen by the analyst.

Electronic data files and the Leskie case
I would like to examine this case more extensively, both for what it tells us about contamination and what it tells us about electronic data files. Jaidyn Leskie was a young boy in Australia who was murdered n 1997. DNA from an alleged rape victim, Ms. P, was found on two items of his clothing, a bib and track pants. There is general agreement that this was the result of contamination (http://viewfromwilmington.blogspot.com/2010/01/forensic-dna-contamination.html), despite the fact that contamination did not show up in the negative control (reagent blank or no-template) samples. It is helpful to know that Jaidyn’s body was submerged in water for several months before its discover, and these conditions are not at all conducive to the stability of DNA. None of Jaidyn’s own DNA was found, only Ms. P’s.

Professor Thompson provided a report for the Leskie case (quoted above). Professor Thompson wrote (http://www.bioforensics.com/articles/index.html), “When I review the casework of forensic DNA laboratories in the United States, I typically ask for and receive copies of the electronic data collected by laboratory instruments during DNA testing. These data show the actual results of the DNA tests. By examining these data, an independent expert can check whether the forensic laboratory interpreted its results correctly and can detect a host of potential problems that would not be apparent from examining the laboratory notes. Electronic data would be particularly helpful in evaluating the present cases. Beyond allowing a cross check of the analysts’ interpretations, it would allow a detailed independent assessment of whether there is evidence of low- level contamination in any samples.”

Professor Krane provided two reports; the supplemental report was written after he was given access to the electronic data files. With respect to the negative controls not showing contamination his first report state, “there is no good reason to expect that contamination would uniformly affect all samples if and when it does occur. In much the same way, Salmonella-tainted meat placed on one part of a countertop at the beginning of food preparation does not mean that all food subsequently prepared for the same (or even a later) meal will be similarly tainted – just those items that also come in contact with the same portion of the countertop can be reasonably expected to have a chance of being tainted.” In previous posts, I have noted that the amount of DNA after it has been amplified by the polymerase chanin reaction (PCR) makes it a serious potential contaminant. However, professor Krane explained why the PCR process itself was not likely to be the source of the contamination in this instance. Professors Thompson and Krane both indicate that substrate controls would have been appropriate in this case.

It is Dr. Krane’s second report that is especially pertinent to the utility of access to the electronic data. He wrote, “My subsequent review of the underlying electronic data associated with the testing performed by the VFSC during their investigation of the death of Jaidyn Leskie in particular leaves me even more convinced that the STR-DNA profile match observed between the condom of the rape investigation and the bib in the Jaidyn Leskie abduction investigation is the result of contamination.” He went on to say, “Now that I have had an opportunity to review the electronic data from the Leskie investigation I, like Professor Thompson, do indeed find low levels of signal that are consistent with Ms. P’s DNA profile and lend substantially more credence to the already compelling proposition that she is the source of the DNA associated with the Leskie bib and track pants.” Professor Krane subsequently wrote in response to one of my inquiries that the observation of these peaks “helped strengthen the case that contamination had occurred by several orders of magnitude.” (emphasis added)

Professor Krane noted that there was a systematic difference in the peak heights depending on the software one used and the analysis options one chose: “However, I thought it might also be helpful to consider the output as it was seen by the testing laboratory primarily for two reasons: 1) no-smoothing with the Macintosh versions of the software systematically makes peak heights larger; and 2) the peak heights generated in this way should be more directly comparable with the threshold established by the testing laboratory in the course of their validation studies. As Professor Thompson points out, “Most laboratories establish a peak height threshold for reporting alleles and make it a practice to ignore peaks falling below that threshold.” I, like him, feel that this practice is appropriate but that low-level signals may also contain useful information in investigations such as this one.” It is possible that peaks could fall above the threshold in the hands of one analyst and below it in the hands of another. The sizes of the peaks in the two figures in this report vary from less than one hundred RFUs to many hundreds of RFUs, documenting the fact that profiles arising from contamination can easily be mistaken for samples arising from genuine contact.

Justification for the release of electronic files in another case
An anonymous DNA forensic expert unaffiliated with the present case had this to say in a case from several years ago:
“The laboratory has provided hard copy printouts of the data that do not allow the possibility of reanalysis by an independent expert. Certain parameters have irrevocably been applied to the data; as such it represents an interpretation by the laboratory rather than the original data. Of additional concern is the fact that the documents provided by the laboratory are represented as fully complying with the discovery request.” The expert gave several areas where independent review requires the electronic data files, including.
1. Determination of the threshold. “Not all qualified experts would necessarily apply the same threshold given identical data…The possibility of false inclusions as well as false exclusions exists if real data that happen to reside below an artificial threshold are ignored.”
2. Scale of viewing. “This ability is an important element in determining whether a particular peak represents and authentic DNA fragment.”
3. Proprietary concerns. “The data itself, as the work product of a government laboratory in a criminal matter, is clearly in the public purview.”
“Honest differences of opinion by qualified experts should be welcomed by the Court. This can only be accomplished if the independent expert is provided full and complete discovery by the government.”

Electronic data files and the Knox/Sollecito case
In response to a question, Dan Krane wrote me to say, “I would like to see that data primarily for two reasons: 1) to determine the actual heights and data collection points of all the peaks (for purposes of mixture resolution and the possibility that some are associated with pull up, respectively), and 2) to calculate a limit of detection from the data in the controls so as to determine if the lab was reliably distinguishing between signal and noise in the evidence samples.”

Professor William Thompson wrote (Tarnish on the 'gold standard:' Understanding recent problems in forensic DNA testing. The Champion. 30(1):10-16, January/February 2006), “A key aspect of discovery in DNA cases is the electronic data produced by the computer-controlled genetic analyzers that are currently used to “type” DNA samples. Analysis of the computer files can not only reveal undisclosed problems and support alternative interpretations of the findings, but also, as discussed above, these files can be crucial for detecting instances of scientific fraud, such as that committed by Jacqueline Blake and Sarah Blair.” Jacqueline Blake was an employee of the FBI whose two-year record of contamination-free work was the result of dumping a portion of the negative control down the sink. Sarah Blair repeatedly substituted a file of a contamination-free blank for the actual blank.

I can think of several things I would look for using the electronic data files. The first is what peak threshold was used for the 400+ runs and whether or not it was the same threshold as the knife blade profile. The second is to zoom in on the small peaks in the bra clasp, as Jason Gilder implied. It is conceivable that the identities of the three additional people who contributed DNA to the clasp could be ascertained. The third is to examine the negative control runs for evidence of fraud, as Professor Thompson noted could be done. The negative controls are the most common place for fraud to be detected.

State of Michigan
One exception to the general rule of complete disclosure of the electronic data files was the state of Michigan (http://www.bioforensics.com/articles/Thompson_Champion_Tarnish.pdf). In 2005 the Michigan State police Issued a statement saying that, “it is the position of the Michigan State Police Forensic Science Division that any release of this (sic) data for processing with non-validated parameters is tantamount to evidence tampering.” Thompson refutes this position: “By analogy, if the police disclosed digital photos of the crime scene, it would hardly be ‘data tampering’ for a defense expert to manipulate the digital images in an effort to enhance them or bring specific details into focus…The use of proper analysis parameters might well become an issue if and when the defense decided to offer into evidence the results of its analysis of the electronic data. But to deny access to digital data on grounds that the defense might analyze it improperly eviscerates the right to discovery.” (emphasis added) I will update this post if I am able to learn whether or not the State of Michigan has changed its policy.

Viewpoints on the Italian Justice System

Part XI in a series on the Knox/Sollecito case

The murder of Meredith Kercher has caused the Italian justice system to come under scrutiny. The first is a letter Count Neri Capponi wrote about the Monster of Florence case, and the second are his comments on the Knox/Sollecito case to Judy Bachrach of Vanity Fair. Count Neri Capponi is a judge and a lawyer, and his son Niccolo is an acquaintance of Douglas Preston and Mario Spezi, authors of The Atlantic article and the book The Monster of Florence.

The travesty of justice undergone by Douglas Preston and Mario Spezi (http://www.theatlantic.com/magazine/archive/2006/07/the-monster-of-florence/4981/) is the tip of the iceberg. The Italian judiciary (which includes the public prosecutors) is a branch of the civil service. This particular branch chooses its members, is self-ruling, and is accountable to no one: a state within the state! This body of bureaucrats can be roughly divided into three sections: a large minority, corrupt and affiliated to the former Communist Party; a large section of honest people who are too frightened to stand up to the political minority (which controls the offices of the judiciary); and a minority of brave and honest men with little influence. Political and dishonest judges have an infallible method of silencing or discrediting opponents, political or otherwise. A bogus indictment, the tapping of telephones, the conversations (often doctored) fed to the press to start a smear campaign, a spectacular arrest, prolonged preventive detention under the worst possible conditions, third-degree interrogations, and finally a trial that lasts many years and ends in the acquittal of a ruined man. Spezi was lucky, because the powerful Florentine public prosecutor is no friend of the Perugia prosecutor’s and, I am told, “suggested” that Spezi be freed; the Perugia court, I am told, accepted the “suggestion.”
Count Neri Capponi
Florence, Italy
http://www.theatlantic.com/magazine/archive/2006/10/letters-to-the-editor/5197/

The Italian legal system, ecclesiastical judge Count Neri Capponi informs me, will not work in Amanda’s favor. “Our system stems from the Inquisition and also from medieval law,” he explains. What this means, in effect, he says, is that justice in Italy “is based on the supremacy of the prosecution. This nullifies the fact—written in our constitution by the way—that you’re innocent until proven guilty.
http://www.vanityfair.com/culture/features/2008/06/perugia200806?currentPage=6

Amanda Knox was accused of slander for her allegation of police abuse (http://perugia-shock.blogspot.com/2010/01/new-proceedings-against-amanda-knox.html). Based on Frank Sfarzo’s report, one is skeptical about how objectively her allegations were investigated. From a report prepared by the human rights group EveryOne on police violence in Italy, “When human rights activists report episodes of violence or abuse of power being perpetrated by rogue officers to local or national institutions, a worrying phenomenon nearly always takes place. Instead of collecting precise reports of the episodes in order to investigate and identify those responsible for the abuse, the superiors shut up like a clam, denying without question that such disgraceful acts could have taken place. They assume a threatening tone with the associations and threaten to report them for slander, libel and defamation etc. This attitude, which the leaders of EveryOne themselves have witnessed on several occasions, prevents the rogue officers being isolated and their behaviour discouraged. On the contrary, it makes them feel part of an agency in which they are allowed to act above the law using violence, threats and acts of gratuitous coercion. According to the activists, after reporting misconduct by uniformed police officers towards racial minorities, it is not rare for the activists themselves to be followed by plain clothes policemen or summoned to police stations or headquarters and “advised” not to take any further action.” (thanks to Observer for pointing out this report*)

Commentator Peter Popham has written a couple of articles on the Italian system. Comparing the British and Italian systems Popham wrote (http://www.independent.co.uk/opinion/commentators/peter-popham-will-knox-find-justice-in-perugia-1826533.html), “One of the great virtues of the British judicial system is that, whatever ideas a detective or prosecutor may have about a case, he is not allowed to voice them until the case comes to court. And a very good thing too.

They manage these things differently in Italy, where prosecutors regularly leak their theories to the newspapers, often in extraordinary detail. Reporters compete for the juiciest tit-bits. As a result, by the time the trial comes around, the public already know what they think about a case, and why. This makes miscarriages of justice horribly likely. Take the Perugia murder: Mr Mignini made up his mind about it, and got his theories splashed across the media, in early November 2007. But weeks later forensic evidence led the police to another suspect who had little or no connection to Knox, Sollecito and their African friend. Rudy Guede, unlike the original three, was tied to the crime scene by fingerprints, hand prints and DNA evidence. In a separate trial he has already been convicted of the murder.

When Guede exploded on the scene, the investigators should have torn up their work and started again. But by this time the "guilt" of Knox and Sollecito was so well established in the media and in the public's mind that there was no going back. The jury sitting on the case absorbed all those early reports. As a result, justice may be done in Perugia next week, but I wouldn't bank on it.”

Popham also brought up (http://www.independent.co.uk/news/world/europe/the-big-question-should-italian-justice-be-in-the-dock-over-the-conviction-of-amanda-knox-1836076.html) an aspect of the Italian system that might work in Knox and Sollecito’s favor, “The good news for them is that the appeal is essentially a re-trial: every aspect of the case will be examined afresh. It is very common for convictions at the first trial to be overturned on appeal. A conviction is not considered "definitive" until it is confirmed by the Court of Cassation.” Another commentator has indicated that Knox’s incriminating statement was thrown out before her trial in a way that might not have happened until after her trial in another country.

With respect to the broad latitude given to the prosecutor for his summation, Scott H. Greenfield (http://blog.simplejustice.us/2009/12/05/a-trial-without-evidence.aspx) lamented, “During the summation, the prosecutor told the jury about the things Amanda Knox might have said to Meredith Kercher before the alleged drug-induced orgy that ended with her throat being slashed.

‘You are always behaving like a little saint. Now we will show you. Now we will make you have sex.’

This would be a horrible thing to say, except that it never happened. No one says that such a statement was ever made. But summations in Perugia aren't limited to evidence, as they are here. Rather, this is a permissible indulgence into fantasy, a made up dramatization of what the prosecutors contend might have happened. It's used to inflame the jury. It's what prosecutors try to do everywhere, except that there are no restrictions on such fabrications in Italy. Still, arousing passion gets a far better visceral response that appealing to reason.”

I have a relatively limited number of facts on which to base an evaluation of the Italian justice system. Broadly speaking, it has things to admire and areas for improvements. My comments here should not be construed as a blanket indictment of the Italian justice system.

*update 3/22/10
www.statewatch.org/.../EveryOne%20-%20Report%20Police%20Violence%20in%20Italy_ENG.pdf

Luminol and some of the footprint evidence

Part X in a series on the Knox/Sollecito case

The story surrounding the footprints in the murder of Meredith Kercher is complex, and it may take more than one post to cover it. However, the recent jury’s statement included a reference to the footprint evidence as one indicator of guilt. In this post we will summarize the analyses of both pro-prosecution and pro-defense points of view with respect to a bloody footprint found on a bathmat and the three luminol-positive footprints in the hallway.

Chemistry and forensics of luminol
Luminol was used to visualize some of the footprints. Luminol is typically used as a mixture that includes sodium carbonate and sodium perborate. Luminol reacts with the iron atom in hemoglobin, and also reacts with other substances, such as bleach and fruit juice, or with substances that contain a metal ion that can catalyze the light-producing chemistry. Fruit pulp is rich in certain peroxidase enzymes that have an iron ion that has much in common with the iron in hemoglobin. In hemoglobin and in the peroxidase family of enzymes the iron ion is bound to four nitrogen atoms of an organic molecule called protoporphyrin IX. The combination of iron and protoporphyrin IX is called heme, which is tightly bound to the protein portions of hemoglobin or catalase, respectively. A fifth nitrogen atom from a histidine residue within the protein also coordinates the iron ion (Frey and Hegedus, Enzymatic Reaction Mechanisms, pp. 203-209). Heme is called a prosthetic group, a nonprotein molecule that helps a protein to do its job.

Both hemoglobin and peroxidases are proteins, but only peroxidases are enzymes (biological catalysts). In the luminol reaction however, both hemoglobin and peroxidases are acting catalytically. A paper by Barni et al., “Forensic application of the luminol reaction as a presumptive test for latent blood detection,” Talanta 72 (2007) 896–913, is a review of luminol chemistry and forensics. Hydrogen peroxide oxidizes luminol with the production of light. The iron ion within hemoglobin or other substances is a catalyst; in other words, one molecule of hemoglobin converts many molecules of luminol and hydrogen peroxide to produce 3-aminophthalate. Thus the catalytic behavior of metal ions partially explains the sensitivity of this test. Since photons of light are emitted in this reaction, the process is classified as a chemiluminescent reaction.

This paper discusses whether one can tell whether or not the substance reacting with luminol is really blood:

“Due to the possible presence of these substances at the crime scene, the luminol test must not be considered sufficiently specific to permit an unequivocal identification of blood [15,18,51,88,89]….

Generally visual examination is used when the luminol test is employed in a forensic situation, rather than instrumental detection of the luminescence. An experienced practitioner may distinguish the true blood-catalyzed chemiluminescence from that produced by other substances by the evaluation of parameters observable to the naked eye such as emission intensity, duration and spatial distribution. However this approach may also lead to misinterpretation, due to a subjective, informal and non-quantitative evaluation, for example, because its intensity is qualitatively much weaker than that expected for blood. In other circumstances an emission of similar intensity may be thought to derive from diluted bloodstains and is accepted. Therefore, caution should be exercised when using the test.”

Thus the Luminol test is a presumptive test and should be confirmed by one that is more specific for blood. However (http://www.friendsofamanda.org/luminol.html), “The prosecution's key forensic witness told the court she can tell by looking at a luminol reaction whether it involves blood or something else, but she did not perform any scientific tests to validate this claim.”

Perugia-Shock
The prosecution attributed one hallway footprint to Raffaele Sollecito. Francesco Vinci testified that the luminol footprint in the hallway is a human footprint but one that cannot be attributed to anyone in particular (http://perugia-shock.blogspot.com/2009/09/sollecitos-expert-questions-all.html). The shape of the big toe and the print of the putative second toe were key elements to his conclusions. Frank Sfarzo wrote, “With such limited elements Vinci is only able to rule out Raffaele (or the girls) and to say that the print is compatible with Rudi. And we can only admire his honesty and consistency.”

Additional posts on the footprints by Frank Sfarzo
http://perugia-shock.blogspot.com/2009/05/footprint-is-mr-sollecitos.html
http://perugia-shock.blogspot.com/2008/06/footprints-are-amandas.html
http://perugia-shock.blogspot.com/2009/07/coroner-sarah-gino-accuses.html

Darkness Descending
Now let us turn to the analysis of the footprints that Colonel Luciano Garofano gave to the author of the book Darkness Descending, by Paul Russell and Graham Johnson (Pocket Books, 2009). Colonel Garofano is a well-known forensic scientist who recently retired from the Carabinieri. He was much friendlier to the prosecution’s case than to the defense’s case, and I do not agree with everything that he said in this book, particularly with respect to the footprint on the blue bathmat discussed below.

“The other problem I have is the way the Luminol was applied. The size of the blobs shows that it was not carefully vaporized but squirted. That creates two problems. It dilutes the sample and it dilates the print. We have a print attributed to Sollecito, which matches his foot in the size of the big toe, the width of the metatarsus and the width of the heel, but does not present the characteristic details each of our feet present. The print can be said to be compatible, but not 100 per cent.”

“Now let’s have a look at the prints attributed to Amanda Knox. There’s one in her bedroom facing the exit to the room, and there are two right feet in the corridor walking in the direction of the victim’s room. The same goes here. The method of application of the Luminol is insufficiently subtle to positively identify a foot, but the result can be said to be generally compatible with Amanda Knox’s.

“But I didn’t see who else they compared the prints with. Just Rudy, Amanda, and Raffaele? So we only have a choice between those? We don’t have the footprint of other women or men, as a comparison? Pity.”

Colonel Garofano believed that it is likely that Raffale’s foot made the print on the blue bathmat, but he did not sound certain. However, he did not discuss the fatal criticism that the prosecution’s expert witness made a serious error in the measurement of this print, discussed below. Colonel Garofano did not address the fact that Rudy’s big toe looks nothing like Raffaele’s, and the bathmat print. Nor did he seriously entertain the possibility that the prints are not even blood. He also did not explain why the print attributed to Ms. Knox points toward Meredith’s room and why there is not a full set of prints. These are serious omissions in that it is difficult to picture how the prints could have been made during the commission of the murder.

Science Spheres
Mark Waterbury discussed some of the problems in the footprint evidence in the hallway and elsewhere (http://www.sciencespheres.com/2009/10/methods-of-polizia-pseudoscientificaa.html):
“Luminol glowing footprints were found in a hallway, and some may have been Amanda's, it is hard to know for sure because they were only compared with her feet, and found to be ‘compatible.’ Again, no controls. Meredith, Laura, Filomena, none of the other resident's feet were compared to these footprints. The footprints were tested for blood, and it came out negative. No blood. So, why are they important? Amanda lived there, after all.”

“Amanda's DNA was said to be found in one of these footprints. Did they also test a meter away from the footprints, to see if her DNA was all over the apartment where she lived? No. That would have been another control experiment. Was the DNA actually associated with the footprint, or did it just happen to be there, because the resident's DNA was all over their apartment, as people's DNA usually is? We will never know. They skipped the control experiments, and presented results without any reference.”

With respect to Meredith’s DNA and the luminol-enhanced footprints, I would argue that not finding her DNA is evidence against the proposition that the substance is her blood. However, finding her DNA would be not conclusive evidence that the substance is her blood. That is because there could be non-blood derived biological material from Meredith mixed into the footprint. In other words finding her DNA would have been consistent with blood being responsible for the luminol reaction but would not demand that conclusion.

Perugia Murder File
A powerpoint, Dear-Mr-Marriott-I-Shrunk-the-Black-Kid.pps, authored by Kermit (http://www.perugiamurderfile.org/viewtopic.php?f=5&t=36) attempted to rebut arguments presented at the Friends of Amanda website. The first of these is that the prosecution’s expert witness Mr. Rinaldi, made a serious error in his size measurements. Kermit’s presentation also addresses two of the problems in ascribing the bloody footprint to Mr. Sollecito, the narrowness of the big toe (http://www.friendsofamanda.org/footprint.html) and the appearance of a mark near the big toe. Mr. Guede’s big toe is shorter than his second toe, but Mr. Sollecito’s second toe does not even show up in his footprint. Mr. Guede’s big toe is narrower than Mr. Sollecito’s. According to Kermit, the reason that the bloody imprint of the big toe seems narrower than Mr. Sollecito’s is that the raised tufts of the blue bathmat have picked up more blood than the unraised portions. Likewise, the mark Sollecito’s expert witness Francesco Vinci would ascribe to a second toe (http://perugia-shock.blogspot.com/2009/09/sollecitos-expert-questions-all.html), Kermit claims is Mr. Sollecito’s big toe. Kermit said elsewhere that his reason for writing his powerpoint presentations were to refute the lone wolf theory of the crime, because the prints were of varying sizes.

I do not find Kermit’s arguments about the raised tufts to be convincing. There is a raised tuft of lighter color in between the big toe and the second mark, possibly from Guede’s second toe. If Kermit were correct, that portion of the bathmat should be as dark as the rest. The validity of Kermit’s measurements has also been questioned by two commenters at a forum at James Randi Educational Foundation (http://forums.randi.org/showthread.php?t=161229&page=126).

Defense-friendly blogs
Charlie Wilkes of the website Friends of Amanda presented the argument that the bathmat footprint measurements made by the prosecution’s witness, Lorenzo Rinaldi, contained a critical error. (http://www.friendsofamanda.org/footprint_measurements.html). Mr. Wilkes also discussed the footprints elsewhere (http://knoxarchive.wordpress.com/2009/11/27/manuela-comodi-asks-for-common-sense/). The site Injustice in Perugia gives a detailed account with images of the footprints (http://www.injusticeinperugia.org/footprints.html). These two sites make several telling points, especially with respect to the luminol-positive footprints in the hallway. The footprint L9 (in the knoxarchive numbering scheme) is attributed to Amanda by Rinaldi. Yet is alone, and it points toward, not away from Meredith’s room. L6 and L7 are two right feet, one of which is attributed to Raffaele. The footprint in the hallway attributed to Raffaele look quite indistinct to me. The other one is attributed to no one at all, and one wonders to whom it belongs. None of the footprints tested positive for blood and none were positive for Meredith’s DNA.

None of the three footprints were part of a trail. It is difficult to see how Amanda Knox and Raffaele Sollecito could have removed other footprints (as part of an alleged cleanup) without also cleaning up Rudy Guede’s nearby bloody shoeprints.

Prosecutorial tunnel vision
The failure to obtain reference footprints is disturbingly reminiscent of the lack of reference DNA from Laura or Filomena, the other two flatmates. It suggests that once the investigators locked onto Amanda and Raffaele, they did not reopen their field of vision. Crime journalist Mario Spezi said about the word “compatible,” in Douglas Preston’s book, The Monster of Florence, which covers a serial murderer in that city.

“Compatible, not compatible, and incompatible are the baroque inventions of Italian experts who don't want to take responsibility. Using 'compatible' is a way to avoid admitting they haven't understood anything. Was the bullet in Pacciani's garden inserted into the monster's pistol? 'It is compatible.' Was that laryngeal break inflicted by someone who intended to kill? 'It is compatible.' Was that painting done by a monstrous psychopath? 'It is compatible.'”

“Perhaps yes, perhaps no--in short, we don't know! If the experts are chosen by the investigators, they say their results are 'compatible' with the theories of the prosecution; if they are chosen by the defendants they say that their results are 'compatible' with the theories of the defense. That adjective should be outlawed!”

Conclusions
Luminol was overapplied to the footprints in the hallway, and a full set of reference footprints was not taken. These two points alone make the prosecution’s attribution of two the footprints to Ms. Knox and Mr. Sollecito, respectively, impossible to accept as solid conclusions. The unattributed footprint forces one to ask who else was in the hallway and when. Moreover, the luminol-visualized footprints did not contain Meredith’s DNA and cannot be said to be blood. The failure to obtain a positive test for blood is possibly the most serious problem for the prosecution’s case. Although I do not claim to be an expert in the analysis of footprints, the bathmat footprint looks like a better match to Rudy Guede than to Raffaele Sollecito. The footprints in the hallway may date from a time prior to the murder, or on the following morning. The lack of reference footprints is one more indication that the forensics in this case was not pursued in an entirely objective manner.

An interim evaluation of the evidence against Knox and Sollecito

Part IX in a series on the Knox/Sollecito case

Here is an interim overview of some of the evidence in the murder of Meredith Kercher; individual issues may be covered in greater depth once the reasons for the verdict have been announced. We have already examined some of the problems with the DNA of the knife and bra clasp. Problematic as the LCN DNA of the knife and the weak, mixed DNA of the clasp are, they are the only things that tie Mr. Sollecito to the room and Ms. Knox to a supposed murder weapon. Without them the whole case looks insubstantial.

Forensic evidence
The investigators failed to perform and especially to report the DNA forensics to the usual degree of completeness, and this leaves many questions unanswered. The investigators did not take Filomena’s or Laura’s reference DNA samples, even though they were Meredith’s roommates. Chris Mellas, Amanda’s stepfather, said that despite a court order in the summer of 2009, the investigators failed to disclose the electronic data files of the DNA evidence (called .fsa files) and logs to the defense. The lack of disclosure of evidence concerning the DNA forensics was one of the reasons that the defense essentially moved for a mistrial in the fall, a motion which was denied. According to Barbie Nadeau (http://www.thedailybeast.com/blogs-and-stories/2009-09-14/no-mistrial-for-knox/), “Just as court adjourned in July, it was revealed that the prosecution had held back key evidence from the defense and civil attorneys. On Monday, clutching the Italian constitution, Sollecito’s lead defense attorney, Giulia Bongiorno, told the judge: ‘My client has been denied his right to adequate representation.’”

Although Ms. Nadeau does not indicate the nature of the evidence withheld, the subsequent Johnson/Hampikian open letter of 19 November 2009 noted specifically lack of disclosure of the .fsa files. Because it is difficult to see why the defense would not share such files with Dr. Johnson and Professor Hampikian (or at least make them aware that the files had been finally disclosed), the open letter tends to corroborate Mr. Mellas’ account. In addition, Bob Graham wrote (http://www.dailymail.co.uk/news/article-1233539/Amanda-Knox-victim-anti-American-trial-campaigners-urge-Hillary-Clinton-launch-investigation.html), “Other forensic experts in several countries – including two from Britain – have started to study the DNA results but have delayed giving a verdict until they have received precise details on the methodology used by their Italian counterparts. The failure of the prosecution to provide these details to Knox and Sollecito’s defence teams is one of their central complaints.”

Why are the .fsa files important? Technical bulletin 40-035 from Chromosomal Laboratories, Inc., is a checklist of what the laboratory is expected to provide, and it states, “Copies of all data files used and created in the course of performing tests and analyzing data in this case, including .fsa files, if applicable. These files should include all data necessary to independently reanalyze the raw data.” In response to a question on this subject, Professor Dan Krane wrote, “The biggest concern that I personally have regarding this case is the refusal of the prosecution to provide the defense with a copy of the electronic data that underlies the DNA test results -- that is virtually unheard of world-wide today and it would be especially important to review that data in a case such as this which seems to involve such low level samples.” According to a knowledgeable source, “Each tested sample has its own file. The file contains the full electropherogram trace information along with other information about the testing conditions (e.g., date, time, injection time, voltage, temperature, current, the RFU threshold used by the analyst). If you have the electronic data, you can use the DNA analysis software (GeneScan & Genotyper or GeneMapper ID) to independently analyze the electronic data. That allows you to examine the results as closely as possible (zoom in on the electropherogram to evaluate low-level results) and establish the RFU threshold of your choosing.”

One leaves DNA by many mechanisms including shedding hair and skin. Finding Ms. Knox’s DNA in her own flat is a very unsurprising result, whether or not it happened to be mixed with Meredith’s blood. Her DNA was found in Mr. Sollecito’s flat as well. The lack of their DNA in Meredith’s room on all but the bra clasp is a silence that shouts. In a pretrial hearing, Mr. Sollecito’s lawyers argued that it would have been very unlikely for him to be able to leave DNA on the bra clasp without also leaving it on the bra as well, yet none was found there (http://www.telegraph.co.uk/news/3255101/DNA-on-Meredith-Kerchers-bloodied-bra-was-due-to-lab-contamination-claims-Sollecito.html).

The other biological forensics was also problematic. Luminol was overapplied to one of the footprints, according to Colonel Garofano in the book Darkness Descending, leading to a loss of detail in the image. Moreover, luminol is a presumptive test for blood, one that should be confirmed by further testing. Although some claim to have the ability to tell blood from other substances via luminol alone, this claim is controversial in the forensic literature. Moreover, defense expert witness Sara Gino stated "We were not told that, first of all, the prints were treated with a substance which should have indicated whether they were blood, and the result was very uncertain." In addition, the investigators did not obtain reference footprints from all of the roommates. The temperature of Meredith’s body was taken about two days after death had occurred, making it harder to estimate the time of death.

The investigators could have performed the electronic forensics much more professionally. For example, the investigators damaged three hard drives, Meredith’s, Amanda’s, and one of Raffale’s, (http://www.dailymail.co.uk/news/article-1234298/Amanda-Knox-The-troubling-doubts-Foxy-Knoxys-role-Meredith-Kerchers-murder.html). Amanda’s hard drive is said to have pictures of her and Meredith, evidence that would belie the impression that the two roommates did not get along, but her drive has not yet been salvaged. Her defense team has offered to pay for a different group of experts to attempt to retrieve the information, but this offer was rebuffed. The investigators also inadvertently erased a piece of potential alibi information (http://www.lastampa.it/redazione/cmsSezioni/cronache/200909articoli/47691girata.asp), the time that the file Stardust was accessed (http://perugia-shock.blogspot.com/2009/09/computer-places-knox-and-sollecito-at.html).


Non-forensic evidence
The prosecution would have us believe that a gang rape occurred, despite the fact that this is an unusual occurrence. Ms. Knox and Mr. Guede had met only twice, the two men had never met, and neither Ms. Knox nor Mr. Sollecito communicated with Mr. Guede in any documented way. It strikes me as extraordinarily risky to commit a crime with someone one does not know. Then Ms. Knox and Mr. Sollecito are imagined to have removed evidence linking them to the crime but not evidence linking Mr. Guede to the crime. In addition, the prosecution changed the motive during the trial but did not have evidence to support either motive.

Raffaele’s kitchen knife is too large to have made the bloody outline of the knife on the sheet. It is too large to make two of the three wounds, and any knife could have made the third. There is no reason for Raffaele and Amanda to have transported the knife in the first place. On top of that we are asked to believe that Raffaele and Amanda would not dispose of this knife, even though Rudy would have seen them use it and could rat them out, and even though the other knife was disposed of. We are also asked to believe that they would clean the blade of the knife but not the handle. This whole scenario would be risible if it were in a third-rate mystery novel.

Raffaele and Amanda are said to have faked evidence of the flat’s being burglarized. I do not find the argument that the break-in was staged to be convincing. There is little evidence pointing that way. Filomena’s room was tidy when she left it, but she recalled that when she returned, there was glass on her clothes. This suggests that someone put the clothes on the floor to simulate a break-in, then broke the window in her room. However, the photographic evidence is not definitive, and it is possible to imagine a number of explanations. Suppose the window were broken, and glass ended up on the laptop. Then suppose that the laptop were moved from the desk to the floor, where some of the glass slid off to some clothing and some remained on the computer. Also, Filomena was allowed back into her room to retrieve some items, and it is possible that she inadvertently did something to put glass on the strewn clothes and remembers the room as though she found it that way.

It has been argued that Raffaele called the Carabinieri after the unexpected arrival of the Postal Police, in an attempt to portray himself as a concerned but innocent citizen, then lied and said that his call to the Carabinieri preceded the appearance of the Postal Police (who were returning missing cell phones). The prosecution wants us to believe that Mr. Sollecito lied to the police about when he placed the call, before he was a suspect. The explanation for what probably did happen is complex, but unless one believes that Mr. Sollecito is very stupid, the prosecution’s theory does not make much sense. Attempting to lie about when the call to the Carabinieri would have little upside (he could have said to the postal police that he was just about to call them or the Carabinieri), and has a huge downside. One would generally believe that the police would be meticulous record keepers about the times of calls and other matters. If Mr. Sollecito were tempted to concoct this story, a moment’s thought should have been enough to convince him of the likelihood of being found out. The two defendants did do some dumb things, but I do not believe that they are that stupid.

Ms. Knox’s incriminating statements were thrown out as evidence for the murder trial; therefore, they cannot be used to assess her legal guilt or innocence. However, suppose we consider her incriminating statements against Lumumba in assessing factual innocence and ask why she made them. Her subsequent comments about the interrogation are similar to those made by people who have been pressured into making false confessions. Francesca Bene’s interrogation provides a modest amount of support for what Ms. Knox said, as discussed in a previous post. In addition, we have Douglas Preston’s account of his being interrogated in a foreign language, and his evaluation that his halting answers made him sound like a liar. It is quite possible that Ms. Knox’s interrogators misperceived similar behaviors of hers as evidence that she had something to hide and therefore pressed harder. Moreover, her later statement, “But I've said this many times so as to make myself clear: these things seem unreal to me, like a dream,” implicitly disavows Lumumba’s involvement.

The investigators claim that no undue coercion was used, but Dr. Giobbi said that he heard Amanda screaming. The investigators claim that they did not record the key interrogations with Amanda that night, despite having previously recorded her interviews and phone calls. Furthermore, they lied when they said that Amanda came in to the police headquarters of her own volition (http://perugia-shock.blogspot.com/2009/05/giobbi-i-gave-order.html). Therefore, Ms. Knox’s account of her interrogation is more credible than theirs is.

Ms. Knox’s allegedly performing a cartwheel at the police station was the fodder for a great deal of negative publicity. Her own explanation of that episode, as relayed through Chris Mellas to Frank Sfarzo of Perugia-Shock, is that she was stretching to relieve stress when a policeman commented on her flexibility. They fell into a conversation, and he asked her what other gymnastics or exercises she knew. Ms. Knox’s testimony at her trial mentioned meeting the policeman but did not say whether or not he asked her to demonstrate other gymnastic moves. Elsewhere, she implied that she did at least some exercises or stretches of her own accord. Therefore, her testimony neither contradicts nor confirms the version Mr. Sfarzo reported. In any case, her yoga or gymnastics have little or no bearing on her guilt or innocence.

What about Raffaele’s probable lie about his cooking with Meredith and his pricking her finger? Does this not generate suspicion? His statement was made only after he had heard that her DNA was found on the knife blade, not before, and it was made without knowledge of the quality of the data. His statement was stupid, but understandable. Therefore, I would say that there is reason to be suspicious of Amanda and Raffaele, probably not enough to indict, and certainly not enough to convict.

The defense have argued that this tragic crime was the work of a lone assailant, Rudy Guede. The evidence linking Mr. Guede to the crime is much stronger; there is a bloody handprint, the bowel movement in the toilet, and there are several pieces of DNA evidence. I am puzzled by the sexual aspect of the crime, but despite this reservation, Mr. Guede acting alone in the rape and murder is the most logical explanation to me. If information about Rudy’s involvement had come back earlier, I doubt that the investigators would have ever moved for an indictment against Raffaele or Amanda. In other words, this is an instance of garden-variety tunnel-vision on the part of the investigators.