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.”
Monday, May 16, 2011
Thursday, March 17, 2011
An analysis of the bra clasp DNA
Part 27 in the Knox/Sollecito case
Update (7 July 2012)
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.
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.
Wednesday, February 23, 2011
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.
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.
Saturday, February 19, 2011
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.
Wednesday, January 19, 2011
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.
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.
Friday, October 22, 2010
The American Bar Association and DNA electronic data files
Part 24 in the Knox/Sollecito case
It is sometimes claimed that the defense teams of Ms. Knox and Mr. Sollecito failed to show up for the DNA forensic testing in this case. Therefore, they do not know what went on and do not have cause to complain about the testing methods or lack of disclosure. This argument is false on a number of fronts. Ms. Knox’s stepfather Chris Mellas said that the defense was given only a few hours of notification, yet Rome is a considerable drive from Perugia (in addition to the defense having other responsibilities). This argument also ignores the fact that several of the experts from the defense only came aboard long after the testing was over. It is absurd to contend that they should be denied free access to the data. However, another problem with this argument is that it assumes that observing the testing is really that critical in the first place.
I posed the question of whether it would be more useful to observe the testing or to have the electronic data files to Professor Dan Krane, one of the cosigners of the open letter on the DNA forensics of this case. He responded:
“Having the electronic data for review is enormously important. Having the opportunity to witness the testing of samples is of marginal utility at best. Reviews of the underlying data for DNA tests often reveals alternative interpretations of the evidence samples, especially in circumstances were small amounts of DNA are involved and it is difficult to distinguish between signal, noise, and technical artifacts. Observing testing rarely provides any more insights than what should be possible from a review of contemporaneous notes that should be part of a lab's case file. Witnessing testing is far from a cure-all. Problems such as contamination of samples can easily arise before a sample arrives in a laboratory yet could not be detected by an expert observing the testing process itself.”
The subject of the electronic data files has been a major theme of this blog’s coverage of the Knox/Sollecito case. Every expert whom I have contacted has spoken in support of full release, and some private DNA forensic companies even have a standard form for the defense to fill out to obtain them. Another benefit of full release of all case files is that serious clerical errors are occasionally found and corrected.
In addition to forensic experts, legal experts within the United States support complete disclosure of all pertinent information, including but not limited to the electronic data files. Nothing close to full disclosure happened in this case. 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.” He reported on the prosecution’s response: “Deputy prosecutor Manuela Comodi brushed off the request for all forensic documentation and added: ‘They have everything they need. That is enough.’”
The American Bar Association’s Criminal Justice Section Standards on DNA evidence discusses disclosure in section 4.1:
(a) The prosecutor should be required, within a specified and reasonable time prior to trial, to make available to the defense the following information and material relating to DNA evidence:
(iii) the laboratory case file and case notes;
(iv) a curriculum vitae for each testifying expert and for each person involved in the testing;
(viii) all raw electronic data produced during testing;
(ix) reports of laboratory contamination and other laboratory problems affecting testing procedures or results relevant to the evaluation of the procedures and test results obtained in the case and corrective actions taken in response; and
Obviously the Italian courts are not bound by the ABA’s guidelines. However, the ABA guidelines are additional documentation, as if any were required, that release of the electronic data files is a near-universal norm in most nations. Ms. Comodi indicating that she has the right to decide what documentation is enough is remarkable given the unanimity of opinion on the usefulness of full disclosure among DNA forensic experts.
It is sometimes claimed that the defense teams of Ms. Knox and Mr. Sollecito failed to show up for the DNA forensic testing in this case. Therefore, they do not know what went on and do not have cause to complain about the testing methods or lack of disclosure. This argument is false on a number of fronts. Ms. Knox’s stepfather Chris Mellas said that the defense was given only a few hours of notification, yet Rome is a considerable drive from Perugia (in addition to the defense having other responsibilities). This argument also ignores the fact that several of the experts from the defense only came aboard long after the testing was over. It is absurd to contend that they should be denied free access to the data. However, another problem with this argument is that it assumes that observing the testing is really that critical in the first place.
I posed the question of whether it would be more useful to observe the testing or to have the electronic data files to Professor Dan Krane, one of the cosigners of the open letter on the DNA forensics of this case. He responded:
“Having the electronic data for review is enormously important. Having the opportunity to witness the testing of samples is of marginal utility at best. Reviews of the underlying data for DNA tests often reveals alternative interpretations of the evidence samples, especially in circumstances were small amounts of DNA are involved and it is difficult to distinguish between signal, noise, and technical artifacts. Observing testing rarely provides any more insights than what should be possible from a review of contemporaneous notes that should be part of a lab's case file. Witnessing testing is far from a cure-all. Problems such as contamination of samples can easily arise before a sample arrives in a laboratory yet could not be detected by an expert observing the testing process itself.”
The subject of the electronic data files has been a major theme of this blog’s coverage of the Knox/Sollecito case. Every expert whom I have contacted has spoken in support of full release, and some private DNA forensic companies even have a standard form for the defense to fill out to obtain them. Another benefit of full release of all case files is that serious clerical errors are occasionally found and corrected.
In addition to forensic experts, legal experts within the United States support complete disclosure of all pertinent information, including but not limited to the electronic data files. Nothing close to full disclosure happened in this case. 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.” He reported on the prosecution’s response: “Deputy prosecutor Manuela Comodi brushed off the request for all forensic documentation and added: ‘They have everything they need. That is enough.’”
The American Bar Association’s Criminal Justice Section Standards on DNA evidence discusses disclosure in section 4.1:
(a) The prosecutor should be required, within a specified and reasonable time prior to trial, to make available to the defense the following information and material relating to DNA evidence:
(iii) the laboratory case file and case notes;
(iv) a curriculum vitae for each testifying expert and for each person involved in the testing;
(viii) all raw electronic data produced during testing;
(ix) reports of laboratory contamination and other laboratory problems affecting testing procedures or results relevant to the evaluation of the procedures and test results obtained in the case and corrective actions taken in response; and
Obviously the Italian courts are not bound by the ABA’s guidelines. However, the ABA guidelines are additional documentation, as if any were required, that release of the electronic data files is a near-universal norm in most nations. Ms. Comodi indicating that she has the right to decide what documentation is enough is remarkable given the unanimity of opinion on the usefulness of full disclosure among DNA forensic experts.
Monday, September 27, 2010
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.
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.
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