Unresolved issues in the latest round of DNA testing in the Hank Skinner case

DNA testing in the Hank Skinner case began again in 2012 after the state of Texas dropped its objections after a fight that lasted over a decade.  After the release of some preliminary data, the prosecution declared that it confirmed Skinner’s guilt, as reported by Jordan Smith in the Austin Chronicle.  Mr. Skinner’s attorney issued a response stating that not all testing was complete and that defense experts had not had a chance to examine the results yet.  Skinner had been found guilty of murdering his live-in girlfriend Twila Busby and her two grown sons.  He claims that he is innocent, and there is an alternate suspect in this case, Robert Donnell, who was Twila’s uncle.

Mr. Skinner’s DNA was found on 10 of 40 items tested.  Many of the items tested appear to have been blood, and they come from various locations around the house.  There are profiles that belong to an unknown individual or individuals, including one unknown profile on a knife that has DNA deposited by Mr. Skinner and by Elwin Caler, one of Ms. Busby’s sons.  There is also an unknown profile from a bloodstain in the sons’ bedroom.  The unknown profiles may not be complete, but that is one of several issues that further testing might resolve.

My conclusions at this time are that the advisory paints an incomplete (therefore misleading) picture of the case and is overinterpreting both the previous DNA results and the present ones.  For example, the prosecution’s advisory summarized the testimony of a witness who claimed that Mr. Skinner threatened her, but the witness later recanted, a fact that was ignored.  The advisory failed to mention anything about two bloody handprints and bloody bootprints that have been discussed in one report on this case. "The State tested the bloody handprints against those of Hank Skinner.  Hank was good for the three handprints near the back of the house.  The handprint on the trash bag, and presumably the handprint on the front storm door, belonged to someone else." The existence of the jacket, the handprints that apparently do not match, and the bloody bootprints that are not his size are some of the objections to the prosecution's case.

With respect to DNA profiling the first problem is that the most compelling piece of exculpatory evidence, what one person identified as Donnell’s jacket, has been lost before it could be tested.  The jacket is stained with blood and sweat.  If either Donnell’s DNA were found on the jacket or if some of the blood were Twila’s), that would establish reasonable doubt, at the very least.  However, at present there is no reference profile of Robert Donnell (see also below).  The failure to obtain his profile could be a consequence of the investigatory tunnel vision that has plagued this case, as a previous entry here indicated.  I suggest that the State of Texas exhume his body or obtain his DNA in some other way.

A second problem centers around whether Twila was sexually assaulted on the night of her death.  The vaginal DNA profiling was performed by standard autosomal DNA methods, in which DNA deposited by a possible perpetrator can become overwhelmed by the victim’s own DNA.  There is nothing in the public record of Y-STR testing being performed, despite its routine use in sexual assaults.  The unique advantage of Y-STR profiling is that it involves only the Y chromosome, which women do not have; therefore, a male contribution to a mixture can be selectively amplified by the polymerase chain reaction.  Either the laboratory did not do Y-STR testing, or it did but the results were not included in the advisory.  In addition the fingernail clippings either showed Ms. Busby’s DNA, or they did not show any DNA.  Moreover, fingernail scrapings can also be tested with Y-STR forensics, which has marginally higher sensitivity than autosomal testing.  Mr. Skinner’s attorney indicated that the defense has requested additional testing; therefore, it is possible that Y-STR results will be forthcoming.

A third problem concerns the existence of DNA from at least one unknown person.  The evidence with respect to the knife appears inculpatory at first glance, yet it is undisputed that Mr. Skinner was bleeding that night.  It is likely that his contribution to the knife DNA profile arose from his blood, and one obvious possibility is that he cut himself while committing the murders. The defense should raise two alternate hypotheses.  There is chance that his profile arose from casual use of this knife around the house prior to the murder.  In addition, Mr. Skinner was incoherent according to one witness due to a combination of alcohol and codeine; therefore, his DNA could have been deposited by his possibly handling the knife at some point after the murder when he was allegedly stumbling around the house.  It is also undisputed that he was in close proximity to the victims that night; therefore, positing that he handled the knife, although speculative, is not unreasonable. 

The Skeptical Juror wrote, "Fresh blood drops were found on the sidewalk, near the front door of the house where the murders took place.  Those drops were tested.  The DNA from those drops belonged not to Hank Skinner, but to an unidentified male." The most recent DNA profiling also did not test the gauze, even though it may have been used to wipe the knife, which is odd.  I wonder whether this profile was similar to or different from the ones turned up in the most recent round of testing, and I also wonder who the DNA donor(s) is(are).  The former question might be addressed by treating the DNA from the blood drops on the sidewalk as something like a reference profile.  DNA mixtures are sometimes deconvoluted by a process known as sequential unmasking, in which a forensic worker is given information about the reference profiles in a gradual manner, only after completing a relevant portion of the analysis.  Yet even the mere existence of blood from another person calls for an explanation.  Was the blood fresh?  If so, who was bleeding besides Mr. Skinner, Ms. Busby, Mr. Caler, and Mr. Randy Busby, Twila’s other son (all of whose reference profiles are available)?

Finally, a fourth problem is that one of the samples may have been contaminated with some DNA from a laboratory worker.  This kind of event actually happens not infrequently in DNA profiling, but it does mean that the defense needs to scrutinize the negative controls and machine logs to look for other evidence of contamination.  Negative controls will detect large scale contamination that affects multiple items of evidence, but will often not detect sporadic contamination affecting a single sample, as Professor William Thompson has pointed out:  “However, the same processes that cause detectable errors in some cases can cause undetectable errors in others. If DNA from a suspect is accidentally transferred into a ‘blank’ control sample, it is obvious that something is wrong; if the suspect’s DNA is accidentally transferred into an evidentiary sample, the error is not obvious because there is another explanation — i.e., that the suspect is the source of the evidentiary DNA.”

One hopes and expects that future rounds of DNA testing will address some of these shortcomings.  Both the victims’ families and Hank Skinner deserve nothing less than a full accounting of what happened that terrible night.  However, the prosecutor brings charges in the name of the people; therefore, every citizen has some responsibility for what happens in a courtroom, including the times when there is a miscarriage of justice.

Hank Skinner, the death penalty, and investigative tunnel vision

Update I, 5 November 2011

The Skeptical Juror wrote, "The hairs found clasped in Twila’s hand, the hairs pronounced by the DA to belong to the killer, turned out to come from a male, maternal relative of Twila. They did not come from Hank Skinner. According to the standard set by the DA before the testing, those hairs exonerated Hank Skinner." Twila's uncle had stalked her the night of her murder. The DNA testing described appears to be mitochondrial DNA forensics (one inherits mitochondrial DNA from one's mother). My tentative interpretation of the mitochondrial DNA is that the data tend toward innocence but are not yet conclusive. As others have said, I am not certain Mr. Skinner is innocent, but I cannot see a persuasive reason not to test the items in question.

***********************

The state of Texas is trying to put Hank Skinner to death for the murder of a family of three people. DNA testing showed that blood smears on his shirt matched two of the victims. Mr. Skinner was undoubtedly at the scene of the crime, but his mental and physical state at the time open the question of whether or not he would have been capable of committing murder. A recent Texas law was designed to apply to this case, to allow further DNA testing.

Radley Balko has been following the case of Hank Skinner for some time. Writing for Reason Balko noted, “In 2000 DNA tests were conducted on blood taken from a roll of gauze and a cassette tape found in the house; that blood didn't match Skinner, his girlfriend, or her sons.” At Huffington Post he wrote, “There is DNA from the crime scene that could exonerate Skinner -- or could affirm his guilt -- that has never been tested. That includes blood from the murder weapon, blood from a jacket left in Busby's home, a rape kit taken from Busby, scrapings from under Busby's fingernails and hairs she was clutching at the time of her death -- hairs that likely came from her killer.”

The State of Texas has argued that since Skinner’s attorney at the time did not press for testing these items. Radley Balko continued, "’They only tested the material they thought would implicate Skinner,’ [Professor David] Protess told me in an interview last year. ‘They fixated on their suspect, and once they thought they had enough for a conviction, they stopped.’” Private investigation several years after the crime turned up a second plausible suspect.

Students of the Knox/Sollecito case should be especially troubled by investigatorial tunnel vision. In the first place, there are good reasons to question the competency of Skinner’s first attorney, apart from his decision not to seek testing. Second, his attorney’s decision was contrary to the wishes of Mr. Skinner, as he expressed in a letter in 1994. Third, the whole issue of whether or not the attorney should have sought testing is misdirection: the investigators should have tested these items as a matter of good forensic science. Here is a link to a petition for full DNA testing.

Amanda Knox and Raffaele Sollecito: There is no mixed blood

Part 32 in the Knox/Sollecito case

Introduction
The Conti-Vecchiotti report casts further doubt upon the reliability of the bra clasp and the kitchen knife, two of the strongest, yet seriously flawed, pieces of evidence against Ms. Knox and Mr. Sollecito in this case. With the appeals trial expect to wrap up shortly, it is time to reexamine the strength of the other biological evidence against them, especially the mixed DNA traces. An anonymous pro-guilt blogger did so several months ago. The related question of whether or not the luminol-positive areas (some of which had DNA profiles) were truly blood was the subject of a previous entry here and a discussion at Let’s Talk about True Crime.

Which samples contained mixed DNA?
There are two amorphous, luminol-positive spots in Filomena’s room (Reps. 176 and 177), although the alleles which may be Amanda’s are quite weak. There is a shoe print in the hallway (Rep. 183). In the shared bathroom there is a sample from the bidet drain, the cotton flock container on the floor, and from the inside of the washbasin (Rep. 137). There is no DNA from Amanda in the murder room, either by itself or mixed with Meredith’s DNA.

Did the jury in the first trial find the mixed DNA to be significant?
Barbie Nadeau wrote, "The defense's biggest mistake, according to interviews with jurors after the trial, was doing nothing to refute the mixed-blood evidence beyond noting that it is common to find mingled DNA when two people live in the same house. The jurors needed more than that. 'To have mixed blood, you have to both be bleeding,' one of them remarked to me after the verdict. It was obvious that Meredith was bleeding, but why was Amanda bleeding?'" ("Angel Face," pages 152-153)

Does mixed DNA indicate mixed blood?
The passage quoted above implies that at least one juror thought that the two were equivalent, but this is a fallacy. There are three possibilities with respect to mixed DNA and blood: neither source, one source, or both sources of DNA could be blood. A commonly repeated fallacy of this case is to equate a mixture of DNA with a mixture of blood (to assume possibility three). For example in a recent Dateline broadcast Barbie Nadeau said, “I have to say, I live in a house with 3 people, my two sons and my husband, I guarantee you I have no mixed blood with any of them, anywhere in my house. I don't bleed where they bleed, we don't bleed at the same time. There would never be my mixed blood, their blood and my blood anywhere ever.”

The false equivalence between mixed DNA and mixed blood dates back at least to the publication of the book Darkness Descending. In this book Colonel Luciano Garofano, a retired officer of the Carabinieri, said, “However, here is the electropherogram and you can see that the RFU value is very high, so the sample is undoubtedly blood, which is the body fluid that provides the greatest amount of DNA. In some cases you see higher peaks of Amanda's DNA than Meredith's. Amanda has been bleeding. Nor is it old blood, as the defence might say, because blood decays fast. We have the same result on the cotton-bud box. The light switch was over-scrubbed, but from the film the way the cotton-bud was good enough. There too we have mixed blood. So that's pretty significant for Amanda, Unfortunately for her, she bled at the same time Meredith was bleeding. That's a lot to explain." (Darkness Descending, page 371).

There are several reasons why Garofano’s interpretations are wrong. In response to a question of mine, Professor Dan Krane wrote, “Inferring tissue source from peak heights is just plain silly -- to the point of being absolutely outrageous. It hardly bears more comment than that, but if high peaks mean blood then what would you expect from semen which has a ten to one hundred fold higher concentration of DNA?” Professor Greg Hampikian concurred with the view that peak heights were not an indication of whether or not blood was the source of DNA. The peak heights for Raffaele’s profile on the cigarette butt were reported to me as being about the same height as those on the cotton box, and the former are presumably from saliva.

Colonel Garofano’s claim that the DNA from blood decays quickly is difficult to evaluate. A paper (Park et al., “Direct STR Amplification from Whole Blood and Blood- or Saliva-Spotted FTA without DNA Purification,” J. Forensic Sci., March 2008, Vol. 53, No. 2, 335-41) showed that 1-2 year-old blood samples gave strong signals in DNA profiling when stored in the form of FTA cards (which contain stabilizers); therefore, their study does not exactly refute what Colonel Garofano claimed, but it does not support his claim, either. However, this paper also showed that saliva gave tall peaks in DNA profiling, which is one more indication that peak height cannot be used to infer the biological origin of a sample. The rate of decay of a DNA sample depends upon so many factors that dating DNA by its degradation is not practical. Furthermore, even if one were to accept that DNA peaks from blood did degrade very quickly, one might have to conclude that the luminol-positive, mixed-DNA samples were not blood, inasmuch as the luminol was applied on 18 December, more than a month and a half after the crime.

What did Massei conclude with respect to the mixed DNA samples?
In contrast to the juror quoted above, the Massei report did not assume that mixed DNA was equivalent to mixed blood (pp. 278-279, English Translation). “It should then be highlighted that in that same bathroom various [300] trace specimens were found, of a mixed nature and testing positively for blood. It is true that, according to what was asserted and explained, it is not possible with a mixed trace specimen that tested positive for human blood to determine which of the trace’s contributors the blood belongs to. In this case, however, non-mixed traces were also found, which were shown to be of a haematological nature [i.e. blood] and turn out to have the biological profile of the victim.” The report continued (p. 279), “And it is probable - not necessary, but probable - that during the following act of scrubbing the hands to remove the blood, he/she left the mixed trace consisting of Meredith’s blood and of cells which had been removed by rubbing during the act of washing.”

The Massei motivations report acknowledged that Amanda had no wounds and therefore was not bleeding. It also noted that DNA by itself gives no indication of when it was deposited (see below). In summary Massei thought that the mixed DNA did not necessarily indicate mixed blood, but he believed that the traces were deposited simultaneously, at least partially on the basis of Amanda’s declaring that the bathroom was clean on the afternoon of 1 November. Such a position is problematic in that a clean bathroom does not necessarily imply a DNA-free bathroom. Moreover, there is no reason to rule out Amanda’s depositing the DNA on the morning after the crime in addition to the possibility that she deposited it before the crime.

Does mixed DNA have to be deposited at the same time?
In general the presence of DNA almost never gives an indication of how or when it was deposited. The abstract of an article (“DNA profiling of trace DNA recovered from bedding,” Forensic Science International, Volume 159, Issue 1, 25 May 2006, Pages 21-26) on DNA profiling states in part: “The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples.” This is a good example of mixed DNA that could not have been deposited simultaneously.

How common is mixed DNA?
Head of the US National Institute of Standards and Technology's genetics group, “[John] Butler has reviewed more than 5000 DNA samples from 14 US labs and found that mixing is a common occurrence: 34 per cent of the samples he studied included DNA from two people, while 11 per cent were three or four-person mixtures.” Although some fraction of the two-person samples are from the victim and the perpretrator, it is not reasonable to suppose that this is the case for all of them, let alone the three or four-person mixtures.

Are there other mixed DNA samples in this case?
In addition to the mixed DNA of Meredith and Amanda at the girls’ flat, there are also three mixed DNA samples containing Amanda’s and Raffaele’s DNA at his flat. One was found in Sollecito’s bathroom, one was found in his bedroom, and one was found on a pair of rubber gloves. The former two are also luminol-positive, but the identity of the luminol-reactive substance is not known. All three mixed samples are likely to be the result of cohabitation. Amanda’s and Raffaele’s DNA was also found on a cigarette butt at the girls’ flat (p. 193, Massei Report, English translation). The cigarette butt is also interesting in that some of the peaks comprising Amanda’s profile are moderate in intensity, despite possibly being the result of secondary DNA transfer (Amanda does not smoke cigarettes).

Do any of the samples contain DNA from a third party?
Sara Gino’s testimony indicated that in sample 177 in Filomena’s room there were alleles besides those of Meredith and Amanda. I have also examined a copy of the electropherogram. In the D19S433 locus, four alleles are marked: 12, 13, 16, and 16.2, but there are unlabeled alleles at 14 and 15 or 15.2. If one acknowledges that it might have been deposited at some other time than the murder, then one must also acknowledge the same possibility for Amanda's DNA.

Could the forensic team have run controls?
Besides the issue of how samples were collected in general, the forensic police could have done substrate controls, where they examined areas for DNA that were a few inches away from putative blood stains, as materials scientist Dr. Mark Waterbury suggested. If they had found Amanda’s DNA in some of those locations, it would have been suggestive of innocent DNA deposition.

They could also have performed or cited studies of DNA deposition in bathrooms, if such studies existed already (I am not aware of any). There are a number of ways that such studies could be performed. For instance, one could take blood from person A and place it in person B’s bathroom, then collect DNA samples. If one found mixed DNA from A and B, it would strengthen the hypothesis that Amanda deposited DNA in the normal course of everyday living.

Is there precedent for mixed DNA arising through contamination?
Many cases of contamination show a mixture of DNA from the analyst and a potential suspect, as discussed in the previous blog entry. One case, the murder of Jane Mixer, showed contamination from two potential suspects, Gary Leiterman and John Ruelas. However, Ruelas was four years old at the time of the murder and lived in a different city. Therefore, this is probably a case in which both profiles arose from contamination.

Could the way the DNA was sampled have resulted in mixed DNA?
The Massei Motivations report (p. 278, English translation) indicates that the defense thought that the mixed traces were meaningless: “All the more so since the samples had been taken using the same blotting paper which had been used for various parts of the bidet and the sink.” Even Colonel Garofano (a strongly pro-prosecution commentator on the case) was dismayed at the way the washbasin trace was collected, noting, “The fact that the sample was collected by wiping both the edge and the plughole is dangerous. You’re likely to find all sorts of stuff in the plughole.” (p. 370, “Darkness Descending”)

Did the police take every precaution to avoid contamination?
No, there are several ways in which the work could have been improved. Ms. Stefanoni’s view was that liquid samples are liable to cross-contamination, but dry traces are not. In the English translation of the Massei report (p. 203) it says that Stefanoni “specified” that gloves were changed “every time an object was touched that was particularly soaked with blood, and when it was obvious that the gloves would be soiled;” On pages 204-205 she indicated that the presence of a liquid is necessary to bring about contamination by touch.

Ms. Stefanoni’s view is out of the mainstream. On page 38 of John Butler's textbook “Forensic DNA Typing,” he wrote, “Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence.” At Forensic Magazine in the article “Evidence Handling and Collection” Dick Warrington wrote, “Go about collecting evidence. I can’t say enough about avoiding cross contamination. Put on gloves, use gloves, change gloves. Do that every time you touch a piece of evidence. Likewise, use disposable tweezers, scalpels, etc. Change these each time they are used, as well.” Warrington also wrote an article for Forensic Magazine called “DNA Collection and Packaging,” that discussed the use of gloves and tweezers to avoid contamination. Orchid Cellmark’s guidelines state, “Use clean latex gloves for collecting each item of evidence. It is recommended the gloves be
changed between the collection of each item of evidence.” If the police handled an item of evidence with Amanda’s DNA then handled an item with Meredith’s DNA, the glove could carry Amanda’s DNA into the other sample.

Are there innocent explanations for the mixed DNA in Filomena’s room?
The luminol work that first identified some of the areas that later were shown to contain mixed DNA traces was performed on December 18, 2007. By this time many police personnel had been in the girls’ flat, and many of Meredith’s items had been tossed about. This raises the possibility that the forensic police tracked the genetic material of either Knox or Kercher into Filomena’s room from the hall. The forensic police who were recorded on 18 December wore one-piece tyvek garments but did not appear to have any outer shoe covering. Former FBI agent Steve Moore noted that they did not change shoe covers going from one room to another that that this creates the potential for cross-contamination. This is especially worrisome in that several members of the team are quite close to the dried bloodstains in Meredith’s room, as can be seen in parts 10 and 11 of a series of videos taken on 18 December 2007.

In addition the luminol-positive spots are only presumptive blood; these tested negative by tetramethylbenzidine, a second type of presumptive test, and there is no record of confirmatory blood testing. Therefore, it is open to debate whether or not the luminol-positive substance is even blood. One photograph of the luminol-positive footprints in the hallway also show blue specks on the ruler and on the boot of one of the forensic police officers. It is unclear what the luminol-positive substance was in this case, or whether it could have contaminated other items of evidence.

Conclusions
Mixed DNA is commonly observed and is not equivalent to mixed blood. In general DNA samples cannot be dated, and any two profiles within a sample may have been deposited at different times. The mixed DNA in the bathroom may have been created by Meredith's blood falling on Amanda's biological matter that was already there. The chances of this happening might have been lessened if the forensic police had taken a smaller trace with respect to the washbasin, for example. Dirty gloves or dropped swabs (which happened elsewhere) made have mixed DNA during collection. The police or the inhabitants of the flat may have tracked Meredith's blood into Filomena's room. The evidentiary value of these mixed DNA samples is very low.

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.

The Independent DNA Experts Weigh In

Part 29 in the Knox/Sollecito case

Judge Hellmann, who is presiding over the appeal, appointed Conti and Carla Vecchiotti as independent experts to review the bra clasp and knife DNA evidence. The translation of their conclusions was provided by komponisto, who also authored “The Amanda Knox Test.” Their report will be discussed in court next month. The formatting (bold or italics) is in the original.


CONCLUSIONS

Based on the considerations explained above, we are able to respond as follows to the inquiries posed at the assignment hearing:

"Having examined the record and conducted such technical investigations as shall be necessary, the Expert Panel shall ascertain:

1. whether it is possible, by means of a new technical analysis, to identify the DNA present on items 165b (bra clasp) and 36 (knife), and to determine the reliability of any such identification"

- The tests that we conducted to determine the presence of blood on item 36 (knife) and item 165B (bra clasps) yielded a negative result.

- The cytomorphological tests on the items did not reveal the presence of cellular material. Some samples of item 36 (knife), in particular sample "H", present granules with a circular/hexagonal characteristic morphology with a cental radial structure. A more detailed microscopic study, together with the consultation of data in the literature, allowed us to ascertain that the structures in question are attributable to granules of starch, thus matter of a vegetable nature.

- The quantification of the extracts obtained from the samples obtained from item 36 (knife) and item 165B (bra clasps), conducted via Real Time PCR, did not reveal the presence of DNA.

- In view of the absence of DNA in the extracts that we obtained, with the agreement of the consultants for the parties, we did not proceed to the subsequent amplification step.

2. "if it is not possible to carry out a new technical analysis, shall evaluate, on the basis of the record, the degree of reliability of the genetic analysis performed by the Scientific Police on the aforementioned items, including with respect to possible contamination."

Having examined the record and the relevant documents, we are able to report the following conclusions regarding the laboratory analyses performed on Item 36 (knife) and Item 165B (bra clasps):

ITEM 36 (KNIFE)

Relative to the genetic analysis performed on trace A (handle of the knife), we agree with the conclusion reached by the Technical Consultant regarding the attribution of the genetic profile obtained from these samples to Amanda Marie Knox.

Relative to trace B (blade of the knife) we find that the technical analyses performed are not reliable for the following reasons:

1. There does not exist evidence which scientifically confirms that trace B (blade of knife) is the product of blood.

2. The electrophoretic profiles exhibited reveal that the sample indicated by the letter B (blade of knife) was a Low Copy Number (LCN) sample, and, as such, all of the precautions indicated by the international scientific community should have been applied.

3. Taking into account that none of the recommendations of the international scientific community relative to the treatment of Low Copy Number (LCN) samples were followed, we do not accept the conclusions regarding the certain attribution of the profile found on trace B (blade of knife) to the victim Meredith Susanna Cara Kercher, since the genetic profile, as obtained, appears unreliable insofar as it is not supported by scientifically validated analysis;

4. International protocols of inspection, collection, and sampling were not followed;

5. It cannot be ruled out that the result obtained from sample B (blade of knife) derives from contamination in some phase of the collection and/or handling and/or analyses performed.


ITEM 165B (BRA CLASPS)

Relative to Item 165B (bra clasps), we find that the technical analysis is not reliable for the following reasons:

1. There does not exist evidence which scientifically confirms the presence of supposed flaking cells on the item;

2. There was an erroneous interpretation of the electrophoretic profile of the autosomic STRs;

3. There was an erroneous interpretation of the electrophoretic profile relative to the Y chromosome;

4. The international protocols for inspection, collection, and sampling of the item were not followed;

5. It cannot be ruled out that the results obtained derive from environmental contamination and/or contamination in some phase of the collection and/or handling of the item.

THE EXPERTS

Prof. Carla Vecchiotti

Prof. Stefano Conti

The Independent DNA Experts and the Electronic Data Files

Part 28 in the Knox/Sollecito case

Update, 13 June 2011

In the story “Knox appeal: DNA experts to request more time” from the AFP on 20 May 2011, Knox lawyer Carlo Dalla Vedova said “The experts asked the forensic police to hand over information essential to their report on the DNA. They still haven't received it and will therefore request a 40 days extension.” He added, “It's not the first time we've asked for the police to hand over this information,” He also said, “But they need the raw data they have asked for from the police to do so. We first asked for it in 2009 and it's still not been handed over.” This ends the debate about whether or not the forensic files were ever released to the defense during the trial of the first instance.
____________________

Judge Hellmann appointed two independent experts to review the DNA forensic evidence in Amanda Knox’s and Raffaele Sollecito’s appeal. Recently, the experts asked for more time, and reports suggested that they did not yet have access to documents the felt were necessary to carry out this task.

According to Candace Dempsey, forensic scientist under whose supervision the tests were carried out, Dr. Patrizia Stefanoni, turned aside this request. She wrote to Judge Hellman, “In reference to the request of acquisition of CD RAW DATA, one is obligated to explain that the information in the form of this file in the sequencer is never an integral part of the technical report, as far as the object being tested by the forensic geneticist, namely the DNA profile, and that it is already reported in the electropherogram printout, connected to the technical report on which all of the useful date and an evaluation of the genetic profile are reported… Finally, the request asked for by the expert consultants relative to the acquisition of the CD RAW DATA appears incomplete in so much as the name of the ‘sample file’ requested was not specified…”

To help me consider Dr. Stefanoni's refusal refusal, I have consulted with DNA forensics professionals Dan Krane and Jason Gilder of Forensic Bioinformatics, and I gratefully acknowledge their help. The continued lack of file release with respect to the DNA profiling of this case has been a recurring theme of this blog.

Her arguments against releasing further information are essentially:
(1) All of the necessary data are already in the paper printouts of the electropherograms.
(2) The request for data files is insufficiently specific.

Let us examine point (1) first. Dr. Stefanoni’s position appears to be the same as it was when Dr. Pascali was refused data, as noted in Raffaele’s appeal. Yet some of the electropherograms only provide the number of repeats, not the peak height for each peak. Peak heights are essential to evaluate peak height imbalance within a locus, which bears on the question of whether or not a sample is in the low-template range, and whether two peaks within a locus belong to the same or to two different individuals. Peak heights can also be used to quantify the severity of degradation when one compares DNA fragments of different lengths. Peak height ratios also help one to decide whether or not a small peak is a type of artifact known as a stutter. A careful examination of these small peaks is especially important in helping to judge what other DNA is present on the bra clasp besides Meredith’s and presumably Raffaele’s.

In addition, having the electronic data files allows one to calculate a run-specific limit of detection (Gilder et al., J. Forensic Science, January 2007, 52 (1), 97). This process sets a lower limit on the size of which peaks to accept, based on the amount of noise.

It can also be helpful in detecting a type of artifact known as a pull-up. There are four types of dyes used in DNA profiling, each with a different wavelength (color) of detection. Each dye is ordinarily detected in its own channel. Sometimes a large peak gives a small spurious signal because of bleeding from one channel into another (Butler, Forensic DNA Typing (2005), pp. 336-337; 384). According to Christine Funk and Dr. Simon Ford, “Pull-up can usually be identified through careful analysis of the position of peaks across the color spectrum, but there is a danger that pull-up will go unrecognized, particularly when the result it produces is consistent with what the analyst expected or wanted to find.”

Dan Krane was asked to give his opinion about the release of such files in a separate legal matter. He wrote, “I believe that a defense expert cannot competently evaluate the results of an STR DNA test without having access to the test’s underlying electronic data. In my experience, review of electronic data has often led directly to the discovery of important problems or limitations in the STR testing, or to alternative theories of the evidence, that would not have been apparent based on a review of laboratory reports or other laboratory records… In my opinion, review of the electronic data is as important as review of the laboratory’s written notes…There is no legitimate reason for a laboratory to refuse a defendant’s request to examine the electronic data.” (bolding mine) Finally, this blog has previously noted that the ABA standards explicitly call for release of the electronic data files.

Point (2) is equally difficult to comprehend. Clearly Dr. Stefanoni understands that the electronic data files are being requested, yet apparently wants specific file names. It is difficult to see how the independent scientists would know the file naming convention used in Dr. Stefanoni’s lab. Who does Dr. Stefanoni think can provide the specific file names?

Forensic Bioinformatics has a 10-point standard discovery motion, and point 6 covers files. The material should include:
(6.1) All collection files (such as injection lists and log files for an ABI 310 analysis).
(6.2) All GeneScan® files, including sample files and project files.
(6.3) All Genotyper® files, including templates/macros (see Request 5).
(6.4) All GeneMapper® files, including sample files (.fsa files) and project files (.ser files).
(6.5) If the data you are providing includes files from another case that are not pertinent to the instant case (e.g., sample files from another case included in the same run folder), then please identify those non-pertinent samples by name and laboratory code.

Clearly it is the job of the laboratory that performed the test to provide the file names.

Concluding remarks
The failure of Dr. Stefanoni’s laboratory to provide the data to the independent forensic scientists is a continuation of her refusal to provide them to the defense. There is absolutely no legitimate reason for her to do so. As Dan Krane noted, “It is a fundamental tenet of science that two reasonable experts should be able to independently arrive at the same conclusions after reviewing the same experimental data.”

Why I believe that Amanda Knox and Raffaele Sollecito are innocent

Part 25 in the Knox/Sollecito case

The molehill of evidence
It is the evidence that is not there that is the prosecution’s weakest point, as former FBI agent Steve Moore pointed out. Consider blood spatter, for instance: “t is inconceivable that the person stabbing Meredith was not contaminated by blood spatter. Guede was. Anybody holding Meredith (such as was alleged by the prosecution) would be within the spatter zone. Again; blood on clothes and skin.”

There is no evidence that Amanda or Raffaele had any contact with Rudi Guede on the night of the murder. There is no evidence that they took any drugs other than cannabis. There is no DNA of Amanda’s in the murder room, and the only evidence of Raffaele’s is the highly contested bra clasp. Forensic Engineer Ron Hendry refuted the arguments of the prosecution that the break-in was staged. The difference in the amount of evidence against Guede versus Knox and Sollecito can be likened to a strong versus a weak signal, and only by pretending that the strong evidence is no better than the weak evidence can one come to the conclusion that all three are culpable.

The interrogation on the night of the 5th of November
The police seemed to have prior knowledge of Amanda’s text message to Patrick on the night of the murder, and the police may have known that the two of them met on the afternoon of the 5th. We also know from both Amanda’s contemporaneous statements and those of others that she was tired and scared in the days leading up to this interrogation (Candace Dempsey, Murder in Italy, Chapters 5-8).

There are many instances of people making a combined false accusation and confession, and this is one of them. The interrogation started around 11 PM and produced two statements, one around 1:45 AM and the other around 5:45 AM. She asked whether she needed a lawyer and was told that would only make things worse. Her statement the next morning shows considerable confusion. Also, if she had been completely rational during the interrogation, she would never have accused Patrick, whether she were innocent or guilty. She believed that he was at the bar that night, which should give him a rock-solid alibi. Again, Steve Moore’s comments are extremely useful and thought-provoking: “Why would detectives schedule an interrogation overnight? ...the reason they interrogated Amanda all night was to break her. Not get the truth, not get answers, not make Perugia safer; but to break her so that she would say what they wanted her to say.”

An ordinary kitchen knife, not the murder weapon
The large knife from Raffaele's flat did not match at least one and probably not two of the three major wounds. It did not match the bloody outline of a knife in Meredith’s bedroom. The DNA on the handle from Amanda was probably deposited when she used it for cooking at Raffaele’s flat. Although the arguments are sometimes detailed, Meredith’s DNA on the knife is probably due to contamination in the laboratory itself, but it may have been contaminated during its transport.

Shoeprint and footprint evidence
The police tried to insinuate some of the luminol-positive footprints were due to Knox and Sollecito and that all were set in blood. This attempt was intellectually dishonest. All of the shoeprints matched Guede’s sneakers. The luminol-positive footprints in the hall do not appear to be Meredith’s blood, inasmuch as they did not have Meredith’s DNA. The one bloody footprint in the bathroom looks a little bit more like Guede’s foot than Sollecito’s foot, but attributing it to either person unequivocally is questionable at best.

Cognitive bias and tunnel vision
If they are innocent, then how did they get convicted? Amanda and Raffaele were detained and held without charge before the forensic evidence came back implicating Rudy Guede. The day of their arrest the police paraded them through the old town with lights on and horns blaring. This had only happened one time previously in Perugia, according to the memory of one citizen, when a mafia figure was arrested. By the time Guede was becoming a suspect, a major figure in the Rome police department had put Ms. Knox’s picture in the hallway, right next to the arrest of Bernardo Provenzano.

This was not a conspiracy in the sense of a bunch of people sitting around a big table. I think it is a case where the police and public minister (PM) Mignini had made a bold claim about Knox and Sollecito’s involvement and could not back down. It may have been a situation where the forensic police (especially) felt, “To get along, go along.” In addition, the forensic scientists might have really believed that Knox and Sollecito were guilty and subconsciously tilted their results in that direction. Koppl and Balko wrote, “To the extent that it's possible, evidence should be stripped of all context before being sent to the lab.” Given that Knox and Sollecito were already in custody in a high-profile case before some of the evidence was even collected, it is difficult to see how all cognitive bias could have been avoided.

Poor forensics and lack of discovery
The prosecution’s misrepresenting which tests were or were not done and their withholding of electronic data files underlying the DNA forensics suggests that they know how weak their case really is. But it is remarkable how much they did wrong or did not do at all, for no reason that I can understand. There is a possible semen stain on Meredith’s bed that was never followed up with a confirmatory test. There were three computer hard drives that the police so badly mishandled that recovery of the data has been difficult or not even attempted. The collection of the DNA evidence with dirty gloves that were seldom changed is just plain sloppy. There is evidence of Meredith’s blood mixed with Amanda’s DNA in certain places in the flat, but the prosecution misleadingly implied that the samples were from Knox’s blood. Since Amanda lived at the flat, finding her DNA there is not inculpatory at all.

Exculpatory evidence
Meredith is known to have returned home around 9 PM. Many small pieces of evidence point to an earlier time of death, before 10 PM, than the prosecution indicated, about 11:40 PM. Meredith had left a load of laundry in the washing machine, presumably when she left to visit her friends for dinner. Yet the laundry had not been removed. Meredith was probably still wearing her zippered sweatshirt when she was attacked, the garment she wore when she was walking home on a brisk evening. Meredith unsuccessfully tried to call her ailing mother around 8:56 PM but did not attempt to reach her again. Atypically, Meredith did not send any text messages that evening. There were two calls around 10 PM that the Massei motivation report ascribes dubiously to Meredith playing with her cell phone. These activities may be evidence of her attacker trying to turn the phones off. A different cell tower sent a MMS to her phone at 10:13 PM; this falls short of proof that the cell phone had left her flat by this time, but it is consistent with the phone’s being transported at this time.

Meredith’s stomach content and the lack of any material in the duodenum are difficult to reconcile with the time of her last meal (of pizza, then apple crumble), which started around 6:30 PM or earlier. Her friends watched a movie after eating pizza, and they stopped to eat apple crumble. Raffaele’s appeal argues that Meredith’s stomach contents indicate a much earlier time of death, by roughly two hours, to about 9:30 or so. Some argue that TOD cannot be accurately determined by stomach contents alone, but that argument can be taken too far: it is known that Meredith was alive until at least 9 PM. In lieu of a lengthy discussion of physiology, let us take a simple path. Patients are advised not to eat for six to eight hours prior to surgery involving intravenous anesthesia. This avoids the possibility that the patient will vomit and aspirate stomach contents into the lungs. Why would anesthesiology guidelines suggest six hours for a small meal if the stomach remained full after 5 to 5.5 hours?

If her death occurred before 10 PM, then it is somewhere between unlikely and impossible that Amanda and Raffaele are guilty. They were known to be acting normally as of about 8:45 PM, when Ms. Popovic came over. Raffaele’s appeal argues that there was some computer activity long after this time, and the police who examined his computer may have not been experienced with MacIntosh products enough to know where to look. Even if the undisputed computer activity ended around 9:10 PM, it is extremely difficult to see how the two of them had time to get so messed up that they lost control enough to murder someone before 10 PM.

Final thoughts
The appeals process has begun with an examination of the DNA forensics of the kitchen knife and the bra clasp. If the computer evidence is reexamined and the stomach contents are reevaluated, Amanda Knox and Raffaele Sollecito have a reasonable chance of exoneration as a result of their appeal.

An overview of some DNA evidence in the murder of Meredith Kercher

Part 23 in the Knox/Sollecito case

Executive summary
The most severe problems with the bra clasp are that three other partial profiles are present, that it was moved by unknown means before it was collected, that it was handled way too much during its collection, and that it might be within LCN range. Massei’s reasoning with respect to the disputed loci is fundamentally flawed. The most severe problems with Meredith’s profile on the knife are lack of blood, the low peak heights, the alleles that dropped in and dropped out, and the fact that an inferior version of low copy number (LCN) DNA analysis was used. Amanda’s DNA found with Meredith’s blood is not inculpatory, because DNA in one’s home is rarely out of the ordinary, and there are collection problems with these samples. Finally, the electronic data files were not released, and this has hampered the ability of the defense to challenge the evidence properly.

General defense arguments
Besides the argument that a profile is insufficient to identify a person (see below), an argument that the defense can use is that the DNA is present due to innocent activity. For example, Amanda’s DNA on the handle of the knife may be due to her using it to cook. In addition, the defense can argue three things with respect to how a defendant’s DNA came to be on an item of evidence: secondary/tertiary transfer, contamination, and evidence tampering.

The legal standard for a DNA profile and low copy number DNA
One argument that the defense can use is to say that a given DNA profile fails to clear the legal bar for identifying someone. One might use the analogy to a partial fingerprint. An example here is the necessity of testing low copy number (LCN) DNA twice, whereas the knife was only tested once. The bra clasp falls into a gray area in terms the amount of Raffaele’s DNA present; the defense is arguing that it, too, falls into the LCN range. Meredith’s profile on the knife also shows other evidence of being in the LCN range, such as peak height imbalance. Moreover, the majority of the peaks are below 50 RFU in peak height, most labs have set a threshold of at least 50 RFU as the minimum height for a peak to be counted. Meredith’s DNA profile should not have been accepted by the court as evidence, and Raffaele’s lawyers may have success with their argument.

Contamination
If we only allowed the contamination argument in cases where the defense could demonstrate the exact mechanism of when and how it happened, we would exclude known cases where it did happen. The prosecution must perform negative controls under the same conditions as the evidence and disclose the results of these control experiments to the defense. If DNA shows up in the negative controls, my understanding is that all evidentiary samples processed at the same time must be performed over. When no rational explanation for the presence of DNA on an item can be offered, one is forced to assume that the DNA arrived via contamination. Some cases of DNA contamination are the Jaidyn Leskie murder, the Jane Mixer murder, the Farah Jama rape case, the Gregory Turner case, and the profile N case in New Zealand.

The lack of frequent glove changes and the handling of the clasp by many forensic technicians are problematic for the prosecution. Moreover, Dr. Stefanoni’s testimony as presented in the Massei report (pp. 202-203 in the English translation at Perugia Murder FIle) on this subject is open to serious challenge. Her argument is that they did not change gloves with respect to a certain item of evidence and this piece of evidence did not show contamination. Therefore, contamination is difficult, requiring either liquids or vigorous rubbing. This view seems to be at odds with the consensus of the field, as well as the facts of the Gregory Turner case, which involved transfer of DNA from fingernails to a wedding ring.

Confirmation bias
Not doing what amounts to substrate controls on the mixed Knox/Kercher DNA samples might have been due to confirmation bias. Not obtaining DNA reference samples from Laura and Filomena are behaviors consistent with confirmation bias. Using a lower peak height threshold for the knife than any other piece of evidence contradicts the words of an introductory textbook on DNA forensics as well as general scientific principles. However, there is an additional reason to suspect that some form of investigator bias was at work with respect to the knife profile. Sample 164 was blood from the wall of a bedroom, but it was not tested because of a “negative preliminary (quantification) result.” One surmises that there was not enough DNA to continue the forensic analysis. Why should testing have been stopped for item 164, which had blood, when it was continued for the knife, which had no blood?

Expert testimony
The prosecution’s witnesses and the defense’s witnesses do not have equal scientific standing. Dr. Stefanoni has not published any articles on DNA forensics of which I am aware. On the other hand the nine signers of the open letter (Dr. Johnson, Dr. Hampikian, and the seven co-signers) publish regularly. That is not to say that publication record or academic prestige is everything; there is also variation in the abilities of expert witnesses to convince a jury that they are correct. In this matter the defense may have not fared as well.

The bra clasp
The following discussion assumes that a full DNA profile corresponding to Raffaele Sollecito’s is present, but that does not answer the question of how or when it got there. One problem with the bra clasp as evidence against Raffaele is that his DNA is not found on the bra itself, a point that his lawyers raised in the Micheli preliminary hearing. A more serious problem is that partial profiles of three other people have also been reported.

An answer to the question of how Raffaele’s DNA was deposited on the clasp is that it might have been deposited the same way as three DNA profiles from unknown individuals. To put it another way, if we acknowledge that DNA from three unknown individuals came to be on the clasp innocently, then what makes Raffaele’s DNA different? It is very unlikely that four people handed the bra clasp as part of a sexual assault and murder. Primary transfer before the assault seems equally unlikely; most people fold their own laundry, and someone else folding it would only account for one profile. So the unknown DNA had to arrive either from secondary transfer or from contamination.

One possibility involving secondary transfer involves the towels. The towels that Rudy probably took from the bathroom might have had DNA from anyone who washed his or her hands in the bathroom and used the towels to dry off, including Raffaele, who had cooked there. If the towel were placed over the clasp and stepped on, it could transfer DNA to the clasp. This might also explain the deformation of the clasp. Another possible route of secondary transfer is that whoever moved the clasp before it was collected deposited Raffaele’s DNA (possibly originating from the door).

There is a good deal of misunderstanding involving DNA contamination and the clasp. It is often said that the only item that tested positive for Raffaele’s DNA was a cigarette butt, and so how could contamination occur? There are several problems with this argument. First, one should not equate the DNA that the investigators found with the total amount of DNA Raffaele left at the cottage. The investigators seemed to be focused on blood, as well as Meredith’s body. They were not taking a random sampling of the cottage. Second, there is no reason to exclude contamination from the cigarette butt in the lab, although if they were tested far apart in time, contamination is less likely. Third, Raffaele’s reference sample is a potentially serious source of contamination. In PCR-based DNA forensics, the DNA is amplified very roughly a millionfold in amount. That is why good labs separate the pre-PCR from the post PCR-samples.

The amount of Raffaele’s DNA on the clasp is borderline LCN. If it is judged to be below the LCN cutoff, it would ordinarily have to be tested twice and only those alleles that showed up in both runs should be counted. It is sometimes said that the amount of Raffaele’s DNA was so large as to mean that it could only arise from vigorous rubbing (primary transfer). The fact that the amount of DNA is actually low would seem contradict such an argument. However, it is the premise that is wrong; the DNA profile itself can rarely give an indication of when and how it was deposited. One cannot rule out primary transfer when the amount of DNA is low any more than one can rule out secondary transfer when the amount of DNA.

Raffaele’s appeal with respect to the bra clasp
The discussion above presumes that a good, complete profile was found. However, Dr. Tagliabracci disputed that the profile matched Raffaele’s for at least six of the loci. If Sollecito’s profile were strong and if the bra clasp DNA were not a mixture, there might be fewer opportunities for disagreement between Dr. Tagliabracci and Dr. Stefanoni. Of the six disputed loci from the bra clasp DNA profile, Massei wrote (pp. 296-297 of the Perugia Murder File English translation):

“Consequently, there are apparently a considerable number of loci that are not the subject of dispute, a number which seems to be greater than the number of disputed loci and greater than the number of six loci with reference to which Professor Tagliabracci had previously declared, before the current systems were available‚ it was enough ... we made hypotheses even with six loci‛ (page [319] 103). The circumstance now exposed allows, it was held, the following consideration: if, despite the subjective contribution of the geneticist, the interpretative disagreement regarding the non-compatibility of Raffaele Sollecito’s profile with the loci that had contributed to forming trace 165B involved those loci indicated by Professor Tagliabracci during the course of the hearing and at pages 20 and 21 of the previously mentioned memorandum conclusions, it must be held that, for the greatest number of loci at least, the peaks were so clear and the interpretation so sound that they could not be contested. Consequently, the overall result should be considered fully reliable, even disregarding the repetition of the analysis. It should however be noted that Dr. Stefanoni, during the hearing at which she testified, had offered suitable explanations and answers which this Court considers acceptable.”

Raffaele’s appeal document correctly notes that Massei’s argument about the numbers of disputed and undisputed loci is contrary to the principles of forensic genetics. Let us assume that the data are clear enough to avoid ambiguity and consider the following analogy. Suppose that a winning lottery number is 12497635834, and I have a lottery ticket that is 12497235834. And suppose I claim that since my ticket has 10 out of the 11 numbers identical, I am a winner. That argument makes as much sense as Massei’s does.

But what of Dr. Tagliabracci’s statement that six loci used to be enough to form hypotheses? Suppose that initially a complete profile consisted of six loci. If a person matched all six loci, he or she would not be excluded as the DNA donor to that sample. However, if that person matched at only 5 loci and failed to match at the sixth locus, then he or she would be excluded. Now suppose an improved test with 10 loci became available. Then a person who matched all ten loci would not be excluded, and the number of other people who could also match would be much smaller than in the case with 6 loci. However, a person who matched at 9 loci but failed to match the tenth locus would still be excluded, even though 9 is greater than 6.

Massei must believe that at all six disputed loci, the DNA is Raffaele’s, or at the very least that the results in all six loci are indeterminate (if the latter were true, it would indicate that Raffaele’s DNA constituted a partial profile, not a complete one). Massei does not provide a clear reason for rejecting Dr. Tagliabracci’s assessment in favor of Dr. Stefanoni’s. It is difficult to see why a sentencing report the fails to provide reasons is any better than no sentencing report at all.

The knife profile
The peak heights on the DNA profile culled from the kitchen knife are all below 100 relative fluorescence units (RFU), and most are below 40 RFU. This is below any threshold of which I am aware. What was the harm in using a lower peak threshold? One can argue that it obliges the forensic scientist to use the same threshold for all the samples on the basis of consistency. It is a dollars-to-donuts bet that some evidence of contamination could be found at this atypically low peak threshold among the hundreds of samples run.

There is no detectable blood on the knife. The open letter asserts that if a bloody knife were cleaned, one would remove detectable traces of DNA before detectable traces of blood. If one claims that the DNA arose from other tissue, then I would ask how it is possible to remove blood cells and not other cells. The cleaning problem only grows more severe if one claims that the knife were cleaned with bleach and that traces of bleach were found. Even trace amounts of bleach are known to destroy DNA for forensic profiling.

The profile shows evidence of alleles dropping in and dropping out. In other words there is one allele where Meredith’s profile is weak or absent, and there is one locus with two peaks that are not part of Meredith’s profile. The peaks within each locus are often very uneven (as much as roughly threefold) in peak height, yet they should be approximately the same height in a good profile. These problems are to be expected when DNA is in the low copy number (LCN) range. When DNA falls into such a low range of amounts, forensic scientists generally test it at least twice and accept only those peaks that appear in both runs.

One can argue that LCN profiling should ordinarily be accepted by a court. However, Dr. Stefanoni used an inferior version of LCN DNA profiling, one that has never appeared in the scientific literature. LCN profiling is typically done in specialized buildings, away from the laboratory doing regular profiling. These precautions are necessary because LCN profiling is more prone to contamination than ordinary PCR-based profiling. These precautions were not followed with respect to the knife, and it was only tested once.

Another problem with the knife is that the second officer to have possession of the knife was at Meredith’s cottage just before receiving it. This raises the odds of contamination outside of the lab. Meredith’s profile probably arose through contamination in the laboratory, but contamination during the time it was taken into custody is also a possibility.

The mixed DNA samples
A number of samples that appeared to be blood had both Meredith’s and Amanda’s DNA. Three of the mixed DNA samples were probably blood and three may or may not have been blood. This would be very weak evidence under most conditions. Amanda’s DNA is expected to be in many locations in her own home. The fact that samples were not taken close to the blood (essentially substrate controls) means that one cannot rule out an innocent explanation for their existence. To argue that these samples are inculpatory, one is almost forced to assume that Amanda’s DNA is from her blood. White blood cells contain DNA; therefore, Amanda’s DNA might have arisen from her blood. Yet without characterizing or quantifying the amount of biological material that gave rise to her DNA, there is no reason to believe that the samples must be from blood. No such tests were done.

However, the prosecution has two additional problems with its case. First, Dr. Stefanoni did not change gloves when collecting multiple samples (see above). Therefore, she might have mixed samples herself. Second, at least one of these samples had a third profile in it, from an unknown individual. If this person’s DNA arose from innocent means, there is no reason to exclude the possibility that Amanda’s did also.

The lack of DNA
The lack of Raffaele’s DNA or Amanda’s DNA on Meredith’s body, when Raffaele is thought to have restrained her and Amanda to have throttled her calls into question this part of the prosecution’s narrative. The number of actual instances where DNA was used in strangulation cases where the DNA originated from the victim’s neck, as opposed to the victim’s fingernails, is small. However, some instances of alleged domestic violence cases have used swabbing of bruised or reddened areas on the alleged victim as evidence.

Conclusions
The way that the bra clasp was handled and the lack of a clear chain of custody cast doubt on this piece of evidence. The lack of blood on the knife calls into grave question whether the DNA got there before or after the police took it into evidence, as argued in the Johnson/Hampikian open letter. Secondary transfer is a likely means for Raffaele’s DNA being in the bra clasp, and contamination, either in the lab or during collection, is a likely means for Meredith’s DNA being found on the knife. The mixed DNA samples are virtually meaningless. The single most troubling aspect of the DNA evidence is the lack of full disclosure of the electronic data files and other documentation relating to the DNA forensics. The prosecution is acting as if it had something to hide.

Eighteen claims about Edda Mellas’s interviews

Part 21 in the Knox/Sollecito case

At True Justice for Meredith, The Machine has posted eighteen supposed errors (“Why The Media Are Wrong To Rely On Amanda Knox’s Family For Impartial and Accurate Information”) made by Edda Mellas, Amanda Knox’s mother, in an interview on Larry King Live and in other interviews. I invite readers to examine The Machine’s assertions and evaluate their accuracy. Please add citations where appropriate. I’ll get the ball rolling with my replies to numbers 2, 3, and 8, which are given below the three claims made at TJfM.

False claim 2. “He (Rudy Guede) all of a sudden had money that he didn’t have earlier in the day” Edda Mellas is plucking “facts” out thin air with this claim. No evidence was presented at any court hearing that showed that Rudy Guede suddenly had money that he didn’t have earlier in the day on 1 November 2007.

False claim 3. “There is no murder weapon.”
Judge Massei indicates in the sentencing report that Amanda Knox’s judges concluded that the double DNA knife, the larger of the two indicated by Meredith’s autopsy, is indeed the murder weapon. It is totally compatible with the deep puncture wound in Meredith’s neck, and according to a number of independent forensic experts, it contained Meredith’s DNA on the blade.
False claim 8. Rudy Guede’s DNA was in Meredith’s purse.
Edda Mellas’s claim that Rudy Guede’s DNA was in Meredith’s purse is completely untrue. According to the Micheli report, which was made available to the public in January 2008, Guede’s DNA was found on the zip of Meredith’s purse and not inside.

Responses
2. The Machine’s claims about court hearings may be true, but he or she needs to explain where Rudy Guede, who was jobless, obtained the money for a train ticket to Germany. Because the murder occurred at the end of the day on 1 November, Guede’s actions on 2 November and later are equally relevant to a discussion of whether or not he had more money.

3. The Machine does not list these other supposed experts here but does in claim 13. Dr. Stafanoni’s boss, Dr. Renato Biondo, cannot reasonably be called independent. Amanda’s DNA on the handle is strictly meaningless, since we cannot know when it was deposited. The hypothesis that her DNA was deposited when she cooked with the knife is perfectly reasonable. Meredith’s DNA is highly contested. This banal kitchen knife is not compatible with two of the three wounds, and the third one could have been made with any sharp knife, including the one that made the first two.

8. The Machine makes a distinction without a difference. The fact that Rudy Guede’s DNA was found on the zip of Meredith’s purse makes it parsimonious to assume he took Meredith’s money (see claim 2).

Update 1, 4 August 2010

False claim 13: “The DNA is so insignificant. It’s this tiny spot. It’s not blood.” (minute 2.16 above)
Three independent DNA experts - Dr. Patrizia Stefanoni, Dr. Renato Biondo, and Professor Francesca Torricelli - confirmed that Meredith’s DNA was definitely on the blade of the double DNA. The DNA charts themselves show a clear and unmistakable match. Edda Mellas doesn’t seem to understand that DNA evidence almost always involves only microscopic traces of DNA. Dr. Stefanoni testified at the trial that the DNA on the blade could indeed have come from Meredith’s blood.

Response
The Machine ignores that Meredith’s DNA was in the low copy number (LCN) range, demonstrating that he or she understands less about DNA profiling than Edda Mellas does. First, LCN samples should be tested at least twice, and only those alleles that show up in both runs are should be counted. Second, Stefanoni did not use standard LCN conditions in other ways, such as the number of cycles of the amplification step and working under lighting and air-handling conditions that minimize the possibility of DNA contamination. New methods are typically vetted in peer-reviewed journals before they are accepted. Third, the Machine also ignores the well known possibility of contamination, as well as the fact that contamination is a greater risk for samples in the LCN range.

There are some problems with The Machine’s assertion that there is a “clear and unmistakable match.” First, in locus D3S1358 there are two peaks, each with peak height of about 20 RFU, which are not seen in Meredith’s reference profile. They have 15 and 16 repeats, respectively, but Meredith’s profile has peaks at 14 and 18 repeats. Second, in the D21S11 locus, the knife profile has a peak at 30 repeats, but Meredith’s profile has peaks at 30 and 33.2 repeats. It is possible to explain the two extra and one missing peak as a consequence of the DNA being in the LCN range. Yet, this explanation only underscores the fact that Stefanoni should have used the services of a lab that has proper facilities and experience, instead of making up her own inferior method.

It is now well known that Stefanoni went ahead with the analysis, despite the fact that her instrument read “too low,” meaning that there was not enough DNA to continue. What is less well appreciated is the fact that Stefanoni treated this sample differently from other samples, where she discontinued the analysis under similar circumstances. The Machine ignores Stefanoni’s apparent inconsistency.

Dr. Stefanoni’s assertion that the DNA could have come from Meredith’s blood is extremely dubious. The knife tested negative for blood with tetramethylbenzidine (TMB), and Dr. Elizabeth Johnson and Professor Gregory Hampikian indicated that the test for blood is more sensitive than the test for DNA. In other words, if a bloody knife were cleaned of all traces of blood, all traces of DNA would also be removed. They and the cosigners of an open letter on the DNA evidence in this case publish regularly in the field of DNA forensics. The same cannot be said of Dr. Stefanoni.

Furthermore, The Machine ignores the fact that Stefanoni did not turn over the electronic data files or the machine logs. This failure to abide by the international standards of discovery of DNA evidence means that any claim about the quality of the results is suspect. The defense expert witnesses did not have all of the information that they would need to challenge Stefanoni’s dubious results.


Update 2, 4 August 2010, approximately 10:15 PM

“False claim 14: Amanda Knox and Raffaele Sollecito maintained the same story…And most devastating of all, Sollecito stopped providing Knox with an alibi on 5 November 2007.
Sollecito is STILL nearly three years later refusing to corroborate her alibi.”


The Machine’s claim about Raffaele is exactly false. Raffaele backed up Amanda at their hearing in front of Judge Matteini on 8 November 2007, discussed in Darkness Descending on p. 208.
“Judge Matteini said, ‘There are several points, Mr. Sollecito, that differ between your version of today and your version of events as related on the evening of 5 November just three days ago. Can you explain whether you were with Amanda Knox that evening or not?’
Now it was make-or-break time. Matteini had posed the million-dollar question. The one Mignini had been waiting for.
His pay-off was unexpected, effectively an explosive retraction of his initial confession.
Raffaele said, ‘I’m sorry I told you that crap about not being with Amanda. We were together that evening.’
…But now on the key point of the night in question, he was sticking to her like glue again. Backing her up… ‘I can confirm that I spent the night with Amanda Knox.’”

Murder in Italy, p. 198, summarized his appearance before Judge Matteini by noting that Raffaele got the times wrong, but the events right. “Then the judge asked the Italian student what he did remember, prompting a long, dull discussion about the broken pipe under the sink, which he had showed Amanda, discussing with her the probable cause of the leak, a perennial problem in that flat. He also remembered eating dinner with her, watching a movie, working on his computer, getting tired, and going to sleep. Yes of course they slept together. He just couldn’t remember what time they did each action, because he’d been stoned, in a holiday mood, and not punching a time clock.” Finally in his diary Raffaele recalls Amanda saying to him that if she had not been with him that night, she would also be dead.

It is often said that Raffaele did not support Amanda’s alibi of being with him the night of the murder in his appearance before the Supreme Court in the spring of 2008. It was actually Raffaele’s lawyers who appeared, and one of their jobs was to end Raffaele’s detention without charge by any reasonable legal argument. According to Perugia-Shock (25 April 2008) Raffaele’s lawyers invoked the concept of “erroneous assumption,” rather than “erroneous belief.” If they had said “erroneous belief”, it would mean that Amanda was not with him. But the “erroneous assumption” has to do with making an assumption, not so much with the thing being assumed, and then transferring evidence onto him as a result. His lawyers were saying that evidence that might place Amanda at the scene of the crime should not be used to justify holding Raffaele in custody. Raffaele’s lawyers might have meant that Raffaele obviously could not account for Amanda’s whereabouts after he fell asleep. Thus there is no justification for using his lawyers’ appearance before the Supreme Court as evidence that Raffaele claimed that Amanda was elsewhere.