An analysis of the bra clasp DNA

Part 27 in the Knox/Sollecito case

Update (7 July 2012)

Some time ago I reviewed some passages in the English translation of the Massei report that concern the bra clasp DNA and examined better electropherograms.  I would like to discuss these without revealing the reference profiles of any of the individuals.  The interested reader should also consult the translation of the Conti-Vecchiotti report and this blog.

Stutter peaks

On page 206 Stefanoni used the word “noise” in a different manner from the way a spectroscopist would use it. From page 207, “Thus, where there is an allele which has a certain height and such that the peak just before it has a much smaller height, at most 15% of the first one, then the previous peak should be considered noise, just a by-product of the analysis.” One infers that Stefanoni used the word noise to refer to stutter peaks and possibly to refer to other artifacts such as blobs. Tagliabracci also used the word noise to mean stutter on p. 241.

If an allele is found at 17 repeats, one expects to see a peak at 16 repeats that is less than 15% the intensity of the peak at 15 but rarely does one see a stutter peak at 18 repeats.  The basis of stutter is that the primer and the template DNA strands do not always anneal perfectly in the DNA replication process. It is far more likely that the bulge of one extra repeat unit will occur on the template strand than on the primer strand, and that is why the stutter peak almost always has one repeat unit less than the true allele.

A problem for a forensic DNA scientist is that when a mixture consists of one strong and one weak profile, discriminating between the weak profile and the stutter peaks is very challenging.  John Butler (p. 125) wrote, “Mixture interpretation requires a good understanding of the behavior of stutter products in single source samples.”

Threshold values

On page 207 one reads, “The height which is considered reliable for a peak to be qualified as an allele is equal to 50 RFU, the symbol RFU representing the unit of measure employed for these measurements.” This is a remarkable statement in one respect, inasmuch as if Stefanoni actually adhered to it, about 22 of about 29 peaks attributed to Meredith on the knife profile would fail to be scored. In other words, she did not respect her own minimum threshold value in at least one other experiment.

Interpretations

On pages 208-209, one encounters some strange statements. “She was asked if she had considered that peak, number 13 [This peak is in locus D5S818 and its height is 108 RFU], as an allele or as noise. Dr Stefanoni declared that she had not considered that peak as an allele or as noise… it can't be an allele because it is too low with respect to the main peaks.” This argument does not make sense; either a peak is an artifact or it is real. It is not in the correct position to be stutter, and there is no reason why a true allele can’t be smaller than Meredith’s profile. Moreover Tagliabracci questioned Stefanoni’s interpretation of this allele (pp. 241-242), noting that in locus vWA that she had taken a peak of only 65 RFU as an allele. Stefanoni argued that each peak should be judged on a case-by-case basis (p. 209), including information such as the main peak heights. Yet the main peaks in vWA are 84% as high as the main peaks in D5S818 on average, whereas 65 RFU is only 60% of 108 RFU. In other words what objective criterion Dr. Stefanoni used to reject a peak of 108 RFU and keep a peak of 65 RFU is obscure or nonexistent. Finally, there are other peaks in vWA at 17 and 18 repeats that are not labeled yet the larger of the two is about 50 RFU. It is difficult to see why this peak was not considered an allele.

Stefanoni was asked about an alternate interpretation, one in which a minor contributor would have the alleles 12 and 13. “In response to this question-observation, Dr Stefanoni explained that in this case, it would not be possible to explain the Y chromosome, and thus reaffirmed the correctness of the interpretation she had given.” It sounds as if Stefanoni used her attribution of the Y-chromosome profile to Raffaele to then interpret the autosomal DNA as having his profile.

Locus D21S11 is problematic (pp. 241-242). Stefanoni counted as stutter a peak that is 15.6% the height of the next peak. That is higher than the 15% cutoff, which is higher than stutter with which I am familiar. But she counted as real a peak that is 17.2% of the next peak, which is not that much of a difference. This peak could constitute half of Raffaele’s profile in this locus, but the other half would fall underneath one of Meredith’s two peaks, which are roughly sixfold higher. Although it is possible that Raffaele’s other allele is present and contributes additional intensity to the second of the two large peaks belonging to Meredith, I don’t see any reason to assume that it must.

There are peaks on the bra clasp electropherogram that are not part of Raffaele’s profile and are not stutter. In reviewing the bra clasp DNA, I am more concerned than I was before that Stefanoni applied a suspect-centered approach, which would be contrary to good forensic practice.

Introduction
A previous post has examined the DNA on the bra clasp, which has a strong profile from the victim, Meredith Kercher. In this post we will examine the bra clasp DNA on the basis of the available information with respect to whether or not Raffaele’s DNA is present. We will also look into the uncertainties surrounding whether or not the DNA of a third party’s DNA is present. However, a complete analysis would require electronic data files, not electropherogram images, and the defense never received these or other files. All references to the Massei Motivations report are given with respect to the English translation available at perugiamurderfile.org. Dr. Adriano Tagliabracci was an expert witness for Raffaele Sollecito’s defense, and his interpretation of the bra clasp DNA profile clashed with Dr. Patrizia Stefanoni’s, the chief prosecution witness with respect to DNA forensics. The bra clasp also figured prominently in an open letter coauthored by two DNA forensic experts and cosigned by seven others.

Executive Summary
Y-chromosomal DNA corresponding to Raffaele’s haplotype was found on the clasp as well as many alleles corresponding to his autosomal DNA. Dr. Tagliabracci’s critique of Dr. Stefanoni’s analysis, that it was suspect-centered, is probably valid but is tangential to the more important questions surrounding the collection of the clasp. The reanalysis of the clasp in the appeal will likely conclude that a partial profile corresponding to Raffaele’s autosomal DNA (but possibly not a full profile) is present. However, there are one or more other contributors as well, apart from Meredith, and the more salient question is how this DNA was deposited on the clasp. There is no reason to suppose that the third person’s DNA necessarily arrived via a different mechanism from Raffaele’s: Secondary transfer, contamination at the crime scene, and evidence-tampering are all more likely than Raffaele’s depositing DNA on this dubious piece of evidence during the murder, but not depositing DNA on the bra itself or leaving any other trace in Meredith’s bedroom.

Analyzing Mixtures of DNA
It is worth reiterating that analyses of DNA mixtures are somewhat subjective. With respect to the John Puckett case, Chris Smith reported: “Mixed samples are another flashpoint in the DNA wars. It can be exceedingly difficult to separate one person’s DNA from another’s, especially in a degraded sample like this, and there is no universally accepted way to interpret the resulting profile. As the eminent British researcher Peter Gill told a conference of his fellow forensic scientists in 2005, ‘If you show 10 colleagues a mixture, you will probably end up with 10 different answers.’ Even Cheng’s supervisor, a combative man named Matt Gabriel, reluctantly admits on the stand that there is no agreed-on protocol for handling mixed samples.” With respect to the Puckett case Michael Bobelaian quoted Professor Dan Krane: “’There is a public perception that DNA profiles are black and white,’ he told me. ‘The reality is that easily in half of all cases—namely, those where the samples are mixed or degraded—there is the potential for subjectivity.’”

An article in the Journal of Forensic Sciences provides good discussion of how to minimize observer effects in DNA forensics. Among the authors are co-signers of the Johnson-Hampikian open letter and one person who has written extensively on the problem of investigator bias. Note that the proposal indicates that the investigator should not have access to the reference samples until quite late into the analysis process. In other words, one wrong way to analyze an evidence sample would be to lay reference sample electropherograms on top of it and look for matches. Overlaying the reference sample onto the evidence electropherogram after the analysis is complete simply to present the data is fine.

Stutter peaks
Individuals vary in the number of short terminal repeats of DNA at various locations in their chromosomes, and this is the basis of DNA profiling. Larger fragments of DNA emerge later from the capillary tube and give rise to peaks in an electropherogram. Each peak corresponds with one allele. Stutter peaks are a type of artifact in the electropherogram, and they most often show up at a position on the time-axis (which is proportional to the size of the DNA fragment) that is one repeat unit shorter than the true allele. They are usually in the vicinity of 5% of the height of the true peak with which they are related. For the purposes of this analysis, we will assume that any peak that is one repeat unit shorter than an allele in Meredith’s profile is likely to be a stutter peak.

From the Massei Report, about page 243: "Regarding locus D7S820, he [Dr. Tagliabracci] revealed that Forensics had interpreted it, recognizing the presence of two alleles, 8 and 11; they had not taken into consideration a peak, low, but still higher than 50 RFU, corresponding to allele 10." To whom does it belong? In locus D16S539, Dr. Tagliabracci believes that there is a peak at the 13 locus. This allele is also not part of Meredith’s or Raffaele’s profile, but it is quite possible that both are stutter peaks.

Other DNA on the clasp
However, there are other small peaks besides the one at 10 in the D7S820 locus, and because of their positions along the time-axis, it is difficult to believe that they are stutters. In locus D19S433 there are alleles at 10 and 14 repeats that are not part of Meredith’s or Raffaele’s profile. In locus vWA there appear to be peaks at 9, 11, and 18, that are not part of these individual’s DNA profiles. In other words, there is probably DNA on the clasp that belongs to someone other than Meredith Kercher or Raffaele Sollecito. Although it is difficult to say how many individuals contributed, there would seem to be at least two. The critical question is how it came to be there, taken up below. Some have tried to claim that Amanda was a third contributor to the bra clasp DNA. However, there can be no doubt that Amanda's complete profile is not on the bra clasp.

The presence of these small peaks raises another question. Many of these small peaks were not marked on the electropherogram of the clasp, suggesting the possibility that Dr. Stefanoni’s lab did not consider them to be real peaks. Some of them appear to be in the range of 100 RFUs. Although they are small relative to typical peaks, they are as large or larger than all of the peaks in Meredith’s profile from the kitchen knife. This forces one to ask whether a consistent peak threshold were used for all samples, and if not, one has to question whether peak thresholds were changed ex post facto, something that seems contrary to basic principles.

Raffaele’s putative DNA on the bra clasp
Raffaele and Meredith shared eleven alleles out of about thirty. Because Meredith’s profile is roughly eightfold more intense than Raffaele’s putative profile, it is very challenging at the very least to know whether Meredith’s DNA alone or Meredith’s plus Raffaele’s DNA contributed to the strong (>1000 RFU) peaks in the profile. For one thing, peaks heights can decrease from left to right across an elecropherogram due to degradation, which attenuates peak heights corresponding to large DNA fragments more than smaller loci. For another, small peaks vary in intensity more than large ones due to noise and stochastic effects. An additional complication is that Raffaele’s profile has a few peaks that would show up at the same position as stutter peaks from Meredith’s profile would.

Let us now turn to the alleles in which Raffaele’s reference profile is distinct from Meredith’s profile. “Professor Tagliabracci then maintained that this suspect-centric method was detectible in Dr. Stefanoni’s report and presentation because, he affirmed, it was a case of forcing the profile obtained … eliminating or leaving out alleles [257] solely for the purpose of making that profile compatible with Raffaele Sollecito’s profile.” (Massei Report translation, p. 241)

The presence of Raffaele’s DNA in YSTR testing strengthens the case that Raffaele’s autosomal DNA is present on the clasp, though in low quantity. One way to take some of Dr. Tagliabracci’s objections into account is to acknowledge that Raffaele’s DNA is present in the low template number range, as discussed in the appendix below.

Some problems in the collection techniques
As noted above the key question is how did Raffaele’s DNA and the DNA of one or more other individuals become deposited onto the clasp. Contamination can happen when an item of evidence is collected at the scene of a crime, as well as when it is tested in the laboratory. In this series of photographs the same crease in a glove of a forensics worker is seen, indicating that this glove was not changed. On page 13 of the book “Angel Face” Barbie Nadeau reported on Dr. Stefanoni’s cross-examination by Raffaele’s lawyer Giulia Bongiorno. Ms. Bongiorno noted that Dr. Stefanoni’s bracelet was seen in the same position above her glove, again pointing to gloves not being changed.

Dr. Stefanoni’s view was that contamination was much less likely with dry traces than with liquids (Massei Report translation, pp. 201-202; 204-205). “Regarding the possibility of transferring exfoliated cells that may be found on a hand or a glove, Dr Stefanoni explained that in the abstract, anything could be transferred, but it remained to be seen in practice. So, with specific reference to exfoliated cells, she stressed that it would be necessary to press down with force or scratch over a surface where these would have to be present (for example, the back of a person)… With reference to the single-use gloves, Dr. Stefanoni specified that they were changed, in the course of the search, every time an object was touched that was particularly soaked with blood, and when it was obvious that the gloves would be soiled; ‘otherwise, if it is just an ordinary object…I can move it, but this does not lead to my DNA remaining, let’s say, attached. It depends on the object.’” (Massei Report translation, pp. 202-203). One infers from her comments that in the absence of blood or obvious dirt or grime, gloves were not changed. Dr. Stefanoni’s views on how frequently gloves should be changed are not shared by any forensic scientists that I can identify. On page 38 of “Forensic DNA Typing,” the most authoritative textbook on the subject of DNA profiling, John Butler wrote, “Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence.” Other guidelines make the same or similar recommendations.

“She [Dr. Stefanoni] confirmed, therefore, that before having touched the clasp with those gloves, the gloves had not touched any other objects, since they had just been put on.” Yet some of the gloves were dirty, including one used to handle the bra clasp. The clasp was handled by at least two people wearing gloves when it should have been handled by one person using a disposable pair of tweezers. The bra clasp was also recovered more than a meter from where it was originally observed and noticeably dusty. In summary the late-collection and subsequent handling of the clasp substantially weaken its evidentiary value, as noted in the Johnson-Hampikian open letter of 19 November 2009.

Dr. Stefanoni’s view that dehydrated traces are very hard to contaminate is also open to question. In the case of Gregory Turner previously discussed on this blog, a forensic worker contaminated fingernail clippings with both her DNA and Mr. Turner’s DNA from a ring, and there is no reason to believe that liquids were involved. Overall, the techniques in Dr. Stefanoni’s laboratory were not as stringent as they might have been, and this raises the chances of contamination, all other things being equal.

The presence of DNA from one or more persons who are not suspects on the clasp is one of the most serious black marks against the bra clasp as evidence of Mr. Sollecito’s involvement in this crime. It is difficult to see how one or more unknown individuals deposited DNA by primary transfer; therefore, secondary transfer and contamination need to be considered carefully as mechanisms by which both Raffaele’s DNA and the DNA of one or more unknown individuals arrived on the clasp.

Appendix
One way to interpret Dr. Tagliabracci’s remarks is that he criticized Dr. Stefanoni for focusing on peaks in the clasp profile that happened to be in Raffaele's reference profile and ignoring peaks that did not. If Dr. Stefanoni did so because she had prior knowledge of Raffaele’s profile, then such analysis is open to question, as indicated above. Dr. Tagliabracci implied that this was a problem. “He pointed out that that there is a significant subjective element in reading the electropherograms. He focused in particular on locus D5S818, in which two principal alleles are present; together with a third peak with a height of 108 RFU; as this is higher than 50 RFU, it should have been considered an allele. Forensics [la Polizia Scientifica] did not, however, consider this to be the case; instead, they considered the 65 RFU peak to be an allele and observed that, in this way, a compatibility with Raffaele Sollecito’s profile resulted, which otherwise would not have been the case (page 59). With reference to this, Professor Tagliabracci repeated that there was a forced interpretation, which was typical of a suspect-centric attitude (page 60).” (Massei Report translation, p. 242) The quotation above only provides the peak height in RFU, not the number of repeats, which sets the location along the horizontal (time) axis. I do not know which peaks are meant.

Dr. Tagliabracci’s approach seems to be more in line with the spirit of recommendations in the 2008 Journal of Forensic Sciences article cited above, although Dr. Stefanoni denied that she used a suspect-centered approach. The Massei report gives at least five instances where Dr. Tagliabracci differed with Dr. Stefanoni with respect to the interpretation of certain loci. Specifically with respect to D5S818, he challenged her interpretation when she took a smaller peak (65 RFU) in preference to a larger one (108 RFU) to be part of a profile. Because other peaks attributed to Raffaele are generally larger, in the range of 200 RFU, this objection needs to be answered.

The Y-STR testing strengthens the case that Raffaele is indeed a contributor to the bra clasp DNA. Yet how can one explain the inequalities in peak heights in the peaks that correspond with his profile? It is possible for DNA belonging to Raffaele to have highly unequal peak heights for several reasons. One possibility is that this would happen when the non-Meredith DNA is in the low template number range. This could lead to large disparities in peak heights in the two alleles within any single locus because of stochastic effects. Dr. Tagliabracci was aware of this issue, as noted in the Massei Report translation, p. 240. Raffaele's putative DNA on the clasp is very weak, less than 200 pg according to his appeal. The exact amount of DNA depends on the details of the calculation. If the low-template number explanation is invoked to explain differences in peak heights, then the standard protocol for dealing with low template DNA is to run the sample at least twice. A retest has yet to be done but may become part of the appeals process.

Comments on the accuracy of the Lifetime movie about Amanda Knox and Raffaele Sollecito

Part 26 in the Knox/Sollecito case

Lifetime premiered a movie on the murder of Meredith Kercher this week, called "Murder on Trial in Italy." I would like to hear everyone’s thoughts and possibly collect them into a summary. ABC and Candace Dempsey have reported on this movie.

February Updates

Eric Volz’s blog has added an interview with Professor Greg Hampikian, one of the two coauthors of the open letter covering the bra clasp and knife. Dr. Hampikian, of Boise State University, is the director of the Idaho Innocence Project. Lifetime is premiering a movie this Monday, “Amanda Knox: Murder on Trial in Italy.” Edda Mellas and Curt Knox, Amanda’s parents, have been indicted for libel. Professor Alan Dershowitz mentioned their indictment in an interview in La Stampa. A translation of what he said is “I love Italy, but in recent times you have made the freedom of expression very weak: the Italian government heavily influences the media and they charged the parents of Amanda Knox, guilty only of having made a public expressing opinions on the process of Perugia, however, tainted by legitimate concerns. On the ground of protection of freedom of the press is not giving Italy a great example.” Update: Another tranlsation can be found here.

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.

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.

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.

Farah Jama and forensic DNA contamination

Part VIII in a series on the Knox/Sollecito case

I have previously posted on DNA contamination issues and subsequently used this post as a starting point to examine the bra clasp and knife as evidence in the murder of Meredith Kercher. I would like to give an additional example of DNA contamintion, that of Farah Jama, an Australian convicted of rape. He spent sixteen months in prison before being released (http://www.heraldsun.com.au/news/man-jailed-for-rape-has-conviction-overtuned-after-revelation-that-dna-evidence-may-have-been-contaminated/story-e6frf7jo-1225807656413).

The twenty year-old Mr. Jama’s DNA profile was found in connection with a possible rape that occurred at a club for those over 28. The woman had no memory of the night. Mr. Jama claimed that he had been reading passages from the Koran to his critically ill father on the evening of the alleged crime. His brother and a friend gave supporting evidence (http://www.theaustralian.com.au/news/features/dna-in-the-dock/story-e6frg6z6-1225809214024).

According to an article in the Herald-Sun on 7 December 2009, ‘The same forensic officer who conducted the tests on the alleged rape victim had done another unrelated test the day before that involved Mr Jama's DNA.” This is the key point; as we have seen before in other cases, when two samples are run at almost the same time, there exists a real possibility for contamination. Another important take-home message is the need to weigh DNA forensics against other evidence (http://www.news.com.au/couriermail/story/0,,26542662-27197,00.html).

The Monster of Florence and the Tragedy in Perugia

Part VII in a series on the Knox/Sollecito case

There are connections between the Knox/Sollecito trial in the murder of Meredith Kercher and the “Monster of Florence” case. The latter was a string of murders mainly in the 1970s and 1980s. A series of couples on lovers’ lanes were shot and the female bodies mutilated. Although several people were suspected, it is unlikely that the true culprit was ever found. Knox prosecutor Guiliano Mignini became involved in the Florence case well after the murders. He and an investigator, Michele Giuttari, were seeking a connection between the Monster case and the death of a certain Dr. Narducci in 1985 (p. 208, The Monster of Florence). Mr. Giuttari believed that the lack of an autopsy was evidence that the body in Dr. Narducci’s grave was not really his. On 6 April 2002 the body was exhumed and confirmed to be that of Dr. Narducci (p. 213). Mr. Giuttari and Mr. Mignini then argued that the bodies had been swapped twice, the second time just before it was exhumed.

Mr. Mignini claimed to believe that Mario Spezi, Mr. Preston’s coauthor for the book The Monster of Florence, planted evidence in the Monster of Florence case. Later he and Mr. Giuttari made Mr. Spezi into a suspect in the murders (p. 239). Mr. Spezi was indicted for obstruction of justice in the Narducci case and held in isolation for five days in 2006. Mr. Preston was himself interrogated by Mr. Mignini, after which he left the country because he was indagato for the crimes of reticence and making false statements (p. 259).

http://www.theatlantic.com/doc/200607/florence-murder/6.

It is a shame that Mr. Mignini harassed Mr. Preston and especially Mr. Spezi. Their theory of the crime and their identification of a suspect made much more sense than the baroque conspiracy theory Mr. Mignini and Mr. Giuttari favored. The Committee to Protect Journalists said that their, “research and interviews with Italian journalists, some of whom asked to remain anonymous for fear of official retaliation, show a pattern of official harassment against Spezi in connection with his investigation of the ‘Monster of Florence’ case.”

“Mignini filed a request with the preliminary investigation judge of Perugia, Marina De Robertis, to invoke a rarely used law under Italy's criminal code to deny Spezi access to a lawyer for five days, Spezi's lawyer Alessandro Traversi told CPJ. The law is typically applied to the most dangerous criminals, yet Judge De Robertis authorized the measure, and for five days Spezi was denied legal counsel and held incommunicado.”

http://cpj.org/2006/04/crime-journalists-imprisonment-raises-alarm.php

“An appeals court that day ordered that Spezi be released immediately, but it did not issue an explanation as to why it overruled the judge who authorized the imprisonment. Spezi, 60, spent 22 days in prison after being taken into custody on April 7.”

http://www.cpj.org/2006/05/italian-court-frees-journalist-held-after-writing.php

Now let us move on to the Knox/Sollecito case, starting with something that happened to Mario Spezi (from the new afterward to Douglas Preston’s book The Monster of Florence, pp. 325-326):

“A few moments later a timid and exceedingly nervous young woman approached.

“I’m a fellow journalist here in Perugia,” she said quietly. Could I speak with you a moment?”

Spezi invited her to sit at his table.

She looked about furtively, as if to check if she were being followed. Then she lit a cigarette with a trembling hand and, stumbling over her words, blurted out, “I hope they don’t see us together.”

“Excuse me, Spezi asked, but who is ‘they’?”

“Them, the police. Mignini’s men.”

“And why can’t we be seen together? What are you afraid of?

“My name is Francesca Bene,” she said all in a rush, “and I work for a small newspaper here, the Giornale dell’Umbria. Last July I made what I thought was a real scoop in the case of Meredith Kercher.” [Francesca tells the story of a drug addict’s suspicious behavior on the night of the murder.]

“Then what happened? Why wasn’t there any follow-up?”

“I’ll tell you what happened. “ Francesca Bene looked around again. “The very day I published that story, I was summoned to the prosecutor’s office and interrogated by Mignini’s men—in particular that big policewoman, the same one who interrogated Amanda Knox.” (The one Amanda says struck her.)” “She’s violent; she scares me.”

“What was there to interrogate you about?” Spezi said. “You say your story was corroborated by many witnesses who went on the record.”

“Of course. But that didn’t stop them from indicting me for the crime of inciting public alarm by publishing false information.”

“But that’s absurd.”

“I was afraid. I’m the only one who works in my family and if I lose my job…I was afraid. So I dropped the story.”

My hunch is that the drug addict in question is unrelated to the murder of Meredith Kercher. However, from this information it should be possible to identify the policewoman who allegedly hit Ms. Knox. This incident also says much about Mr. Mignini’s character and the power of the prosecutors in Italy to intimidate journalists.

With this background in mind, I would like to offer Douglas Preston’s insights from the interview he gave to blogger Candace Dempsey: “One other detail that American readers might like to know: in Italy, prosecutors are firmly in charge. They tell the police what to look for, where to go, what evidence to analyze, what evidence not to analyze. In America, the police work independently and are specifically trained in evidence gathering and criminal investigation. In Italy, the police must do what the prosecutor tells them. As a result, many criminal investigations in Italy are botched by prosecutors who are judges, trained in the law, who have no background in criminal investigation, police work, or forensic science.”

http://blog.seattlepi.com/dempsey/archives/131443.asp

Ms. Dempsey has also written about the connections between this case and the Monster of Florence case. Her highlighting of Preston’s and Spezi’s dissection of the word “compatible,” a word applied to certain forensic evidence in the Knox/Sollecito case, is worth pondering.

http://blog.seattlepi.com/dempsey/archives/140460.asp