Sunday, July 20, 2014
Part 38 in the Knox/Sollecito case
Background on the DNA profiling of Sample 36-I
In overturning the Hellmann-Zanetti acquittal of Knox and Sollecito, the Supreme Court of Cassation (SCC) published a motivations document in 2013 (the previous blog entry critiqued section 12 of the motivations report on genetic investigations). The present entry discusses section 11, another DNA-related portion but one that focused entirely on the retest of Raffaele’s kitchen knife, the subject of two previous entries here. One of the court-appointed independent experts, Dr. Carla Vecchiotti, had found the sample in question (36-I) by swabbing the knife in 2011. Because this amount was far below the recommended amount of DNA for the reagent kit, she decided that the amount was too small (its concentration was estimated to be 5 pg/µL). Pro-guilt commenters sometimes imply that because the defense had the opportunity to object to certain aspects of DNA testing in 2007, that their subsequent requests for the raw forensic data should be turned aside (this argument is problematic on multiple grounds as discussed in the previous blog entry). Yet if we accept that an objection to some aspect of testing should be lodged immediately, then either the prosecution's observers should have objected to not amplifying 36-I in the spring of 2011, or the prosecution should have admitted that it lost the opportunity to do so when it did not.
Why did the Supreme Court of Cassation demand an amplification of the new sample?
In section 11 of their motivations report the CSC wrote,
“This trace was not subjected to genetic analysis – through a decision made by one of the experts, Professor Vecchiotti, alone, without documented prior authorization to that effect by the Court, who had also given a mandate to attribute the DNA on the present findings on the knife and on the bra hook- because the amount was not sufficient to provide a reliable result, amounting to Law (sic) Copy Number. This choice, however, met the subsequent sharing of the group, on the assumption that such a small sample would not have allowed two amplifications necessary for a reliable result (p. 84 of sentence).
“So that when the Procurator General and the civil plaintiffs demanded to complete the examination, strongly as a result of the scientific contribution of Professor Novelli, geneticist of undisputable fame recognized by the same Court (p. 79 sent.) on the availability of equipment able of operate with safety also quantities of less than ten picograms, in the areas of diagnostic character (even on embryos) in which the claim to certainty is certainly no less pressing than that which animates the legal field, the Court rejected that proposal, assuming that the methods to which Prof. Novelli had referred were "in the experimental phase” (p. 84 sent.), thus freely interpreting and misrepresenting the assumption of bias, which in fact was to remember the use of these diagnostic methods in areas in which you can be certain of the result.
“…Especially since the renewal of genetic investigations were requested in 2011, after four years from the initial time and over which the evolution of instrumentation and methods of investigation had marked significant milestones, as was emphasised by the advisor to the Procurator General, Professor Novelli. Just on receipt of the information from the consultant mentioned who - under the constraint of the obligation to truth, spoke of cutting-edge techniques -, the Court fell into a new gross misrepresentation of argument concerning the reliability of the results of investigations carried out assuming no new findings of such remedies, even through developments emerging at a later time, concerning reasonableness of the grounds (Section I, 25.6 .2007, n. 24667).
“…[the survey must] bring to analysis also the newly sampled trace, according to the most accurate and modern “experimental” analytical techniques, under pain of violation of the law for not making a decisive test and the fallout in terms of manifest illogical reasoning (again for obvious incompleteness of the inferential platform, to have overlooked data that is not only important, but crucial), as was correctly pointed out by the public plaintiff.”
Leila Schneps and Coralie Colmez summarized Novelli’s testimony and offered their opinion in the chapter, “The test that wasn’t done,” in their 2013 book Math on Trial (pp. 83-86): "In court on September 5  and supported by expert prosecution witness Giuseppe Novelli, Stefanoni explained that newer generations of DNA analysis kits existed in 2011 that had not been available in 2007, and these new kits could give results on as small as a couple of cells. She wanted a new analysis performed to confirm that her previous work was correct. The prosecution agreed and asked the judge to order the new tests...Judge Hellmann missed a major opportunity to get at the truth.” According to the CSC, techniques that are appropriate for diagnosis in embryology are also appropriate for forensic investigations. There have been advances in technology, and the new tests should take advantage of them. The results will be "decisive."
Not every observer sounded convinced of the probability-based rationale Schneps and Colmez put forward. Professor Daniel H Kaye wrote, “If these experts’ concern — that the original DNA test was simply detecting traces of Kercher's and Sollecito’s DNA that investigators inadvertently transferred to the knife and bra clasp, respectively — then repeating the tests could well continue to detect that DNA — and prove nothing more than the original tests did.” In other words one weakness in the rationale provided by the SCC is that a newer generation of kits cannot circumvent the problem that one is dealing with DNA in the low template range (see below).
Can one equate pre-implantation genetic diagnosis with DNA forensics?
It may be that Professor Novelli left the court with the impression that single-cell PCR was new technology, but single-cell PCR in diagnostics is at least twenty years old. Yet the problem with using diagnostic practices to justify further low template testing on the knife is that it ignores that one is still dealing with low template quantities and all the problems that such analysis has in a forensic setting.
In the article “LCN DNA Analysis: Limitations Prevent 'General Acceptance'” Dr. Angela von Daal wrote, "The use of PCR for analysis of very low levels of DNA in the field of pre-implantation genetic diagnosis (PGD) has been used to justify general acceptance of LCN analysis in the wider scientific community(12). This argument is flawed. PGD analysis is not analogous to LCN analysis for several reasons. PGD uses pristine cellular DNA from a single source, whereas forensic LCN samples are mostly mixtures and are likely in a state of apoptosis. The complexities of profile interpretation issues seen with STR analysis (e.g., stutter) are not an issue for PGD testing(13). However the most significant difference is that the samples derived from the mother, father and embryo are single-source and the parental samples are of known genotype." In other words the connection between pre-implantation genetic diagnosis and low template DNA forensics is tenuous and trying to extrapolate from the former to the latter is dubious at best. Why Professor Novelli chose to ignore the problems in his analogy is something only he can explain.
Did the SCC understand the unique problems of low template DNA profiling?
And the problems of innocent transfer of DNA get more severe as the sample size gets smaller. Sara Gino testified for the defense in the trial of the first instance, and some of what she had to say is pertinent to this issue. From the Massei report (p. 258, English translation): “She reaffirmed that [the risk of] contamination exists, and emphasised that in minimal quantities of DNA there is not necessarily a greater risk of contamination but it was easier to notice the effects of the contamination and be misled (‘...It's not that the risk of contamination is greater; but it is easier to see the contamination...’ page 92).” In response to a question on this subject, Professor Dan Krane expounded on Sarah Gino’s testimony, “There is absolutely no question but that contamination is a much greater problem in LCN cases than conventional DNA testing. The reasons that it is a greater problem are both because it is easier to detect contaminants ([Sarah] Gino's point) and because it is easier to transfer (and to transfer without knowing) smaller amounts of DNA than larger amounts of DNA.”
Proper low template profiling demands that one rework the entire process of DNA forensics, from collection of the evidence all the way through the final step of analysis of the electropherogram. True low copy number LCN profiling requires ultra-clean, dedicated facilities in order to perform it properly, as clearly discussed in nontechnical language in an article in the New Zealand Herald. Without those extra precautions, the odds of contamination are necessarily greater (or else the precautions are a waste of time and money). In a review article in Nanomedicine Professor Novelli and coauthors wrote, “Very few laboratories perform low template DNA typing properly, because it requires dedicated facilities and great experience, although there are several published methods for the interpretation of such profiles [80-82].” This blog has previously discussed the need for proper air handling facilities.
Even so, within the forensic DNA community there are a number of critics of LCN profiling. Dr. Budowle and colleagues, for example, argued that low template analysis should be restricted to identification of bodies and the generation of leads. In other words using the practices in embryology to justify further testing on the knife was predicated on a very weak analogy and one that has been seriously questioned by molecular biologists. Professor Novelli should have known this and should have acknowledged this limitation to the court.
What other forensic issues did the Supreme Court of Cassation ignore?
In forensics it is impossible to interrogate the DNA as to the time or manner of its deposition. With respect to the knife, the DNA may have arrived from secondary or tertiary transfer prior to its collection or during its handling. Regrettably, the knife was removed from its package at the police station, a breach of good practice. The person who repackaged it had previously been to the women’s apartment earlier that day, a second error. In addition, the negative controls have never been produced in the form of raw data or probably any other form.
What did the proponents of the retest expect to learn?
As this blog has repeatedly argued, the DNA from Sample 36B in 2007 was probably from laboratory contamination. Pro-guilt commenters have resisted this interpretation. In a story from 27 April 2013 the BBC’s Ruth Alexander asked Coralie Colmez this question. “’So what this means in the case of the knife in the murder is that if it were tested again, and once again the DNA was Meredith's profile we could be a lot more certain that the DNA on the knife is indeed Meredith's,’ Colmez says.” On the other hand suppose that the test came out differently. “And if the knife were tested again and the DNA did not match Meredith Kercher's profile? That would be good news for Knox and Sollecito, she says.
‘This would mean that this major piece of evidence against them would be discredited.’” In other words either way the results turned out, there would be useful, arguably decisive, new information.
What did the new test actually tell us about the knife?
Andrea Vogt wrote, "The RIS Wednesday deposited their forensic report on trace 36i, a spot of DNA identified (but not earlier tested) on the kitchen knife alleged to be the murder weapon. “Cento Percento” (100 percent) said Major Berti, discussing compatibility. The RIS found that the DNA was compatible with Amanda Knox, and excluded that it was that of Sollecito, Guede or Kercher. The RIS expert was asked only a few questions from attorneys and the judge. The judge asked why the RIS had done two amplications of the DNA and not 3 or 4. Major Berti described that two is considered the minimum number of amplifications necessary, according to today’s forensic standards, doing less (or more) might have diminished the reliability of the results. The judge also asked about the age of the equipment used. Berti responded that the forensic kit used this time has been commercialized since 2010 and available for use since 2011. At one point the judge stopped a line of questioning by Knox’s Rome attorney Carlo Dalla Vedova, who was asking why the RIS described Knox’s DNA as “fluids” when a prior expert had said the trace did not come from blood. Nencini said: That question was not put to the RIS by this court, it was not their job to determine that. The other experts’ reports are in the case files for everyone to read, he noted, adding: “We cannot put words in the mouth of this expert that were said by another expert.”
The presence of Ms. Knox’s DNA on the handle knife has never been in dispute, and the finding of additional DNA on the blade is not inculpatory. A recent review article on trace DNA noted that DNA transfers can occur after an item of evidence is packaged; therefore, the DNA on the blade may have arisen from transfer after the knife was packaged, or from direct transfer: chef’s knives are often gripped both at the handle and the base of the blade. If anything, the result is modest additional exculpatory evidence. If the knife were the murder weapon, it would have Meredith’s blood. Somehow all of Meredith’s blood would have to be removed, yet DNA from Meredith and Amanda would remain, along with starch. A letter to the court from Professor Bruce Budowle indicates that it would be difficult to clean a knife of blood. There was additional DNA in sample 36-I belonging to an unknown person, a finding which further calls into question the hypothesis that it was the murder weapon.
Are the new results exculpatory?
In early 2014 Leila Schneps wrote, “By the third trial, when a new attempt was made to collect DNA from the knife (which had been swabbed again during the appeal trial, though no tests were then conducted) there was no match to Meredith – a result welcomed by Knox's defence team, though it did not in fact impact on the findings of the first trial.” This passage presents quite a contrast with the earlier quote from Coralie Colmez (above).
There are two important issues. The first point is that Meredith Kercher’s DNA was not found, and this was the whole reason for performing the test. The people who argued for the test did so on the basis that the retest might improve the reliability of the 2007 result. They should at the very minimum concede that the original result on the knife has now been “discredited,” as Ms. Colmez wrote. The second point is that the 2013 result did indeed impact on the finding of the first trial. The Carabinieri noted that at least two amplifications of the DNA must be done, and Stefanoni apparently did only one. Therefore, the lack of retesting with respect to the original result is indeed makes it unreliable by the expert testimony of the Carabinieri, which support the testimony of Conti and Vecchiotti. In other words the result and the testimony are a one-two punch that should have knocked the kitchen knife right out of the trial.
Is the Nencini court being impartial and objective with respect to the forensics of this case?
An early report from Andrea Vogt on Judge Nencini’s motivations document from the Florence court indicates that Nencini harshly criticized the rationale for Conti and Vecchiotti’s not completing the test in 2011. For the reasons given above, this argument has no merit. A cynical observer might conclude that it is a canard to distract future courts from the real issue, which is that Meredith Kercher’s DNA was not found, and this was supposed to be a decisive test. Pro-guilt commenters might now argue that the test would have been decisive if Meredith’s DNA had been observed in 36-I but any other result is inconclusive. However, such an argument prompts a question: Why should the Nencini court order a test that might only be beneficial to the prosecution and not also grant requests that are favorable to the defense? This blog has long argued that the failure of the prosecution to release the raw DNA data constitutes a serious breach of discovery, rendering the trial unfair.
Another serious logical problem with the SCC and the Nencini court is the selective indignation about the lack of amplification of 36-I in 2011. Mrs. Stefanoni has failed to produce many electropherograms which showed positive quantification of DNA, some of which may contain highly probative evidence. The defense has every right to see these data under any reasonable standard of discovery. One profoundly wishes that the Nencini court had demanded the production of the electropherograms that originated from a putative semen fraction, to take just one example. The fact that it did not do so is only one reason to call its objectivity into question.
Thursday, September 19, 2013
Part 37 in the Knox/Sollecito case
Part A: Errors in the principles of DNA forensics and discovery
The Italian Court of Cassation, the supreme court of Italy, released a report giving its reasons for overturning Amanda Knox and Raffaele Sollecito's successful 2011 appeal and sending the case for another trial in Florence beginning at the end of September. The report by the Corte Suprema di Cassazione (CSC) has been translated into English twice, and a comprehensive critique has appeared. Page numbers below refer to the first translation. The CSC’s report made a number of highly questionable assertions or omissions with respect to DNA contamination in general and with respect to the evidence in this case:
“It was also ruled out by the same experts that contamination occurred in the laboratory. Professor Novelli said that the origin, the vehicle of contamination must be demonstrated: he specified to have inspected 255 forensic sample extracts at the Polizia scientifica, had analyzed all profiles and did not see any evidence of one single contamination; he excluded absolutely persuasively that the contaminant could be present intermittently and that DNA could remain suspended, and then fall on a particular item.” (p. 67)
“but above all it [the discourse of justification] is based on the erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods tested, the results of which could certainly be called into question…” (p. 69)
The moment of DNA contamination is often not known
Should a mechanism of contamination have to be demonstrated? One problem with this argument is that the mechanism of contamination may only be revealed years after the fact or not at all. In the Jaidyn Leskie case (see below) it was shown that the evidence items were in the same lab closely in time, but the exact moment of contamination was elusive. Modern forensic DNA testing relies upon the polymerase chain reaction (PCR) technique, which produces many copies from a small number of DNA molecules. Dr. Donald Riley wrote, “PCR is also very similar to what happens when a clinical infection occurs. Clinicians have known for many years that a single germ (bacterial cell or virus) contaminating a wound can produce a massive infection. Similarly, a DNA molecule can contaminate (infect) a PCR and become a significant problem. The ability of small amounts of DNA to produce false and misleading results is well-known and well-documented within the research community, where the technology originated. Anyone who has caught a cold from an unknown source, or who has a pollen allergy should have some sense of how easily PCRs are contaminated. Actually, it is probably easier to contaminate a PCR than to catch a cold since unlike our bodies, PCRs lack immune systems. The only protection PCRs have is the technique of the analyst, use of control samples to monitor contaminants and careful interpretation.” Dr. Riley went on to say, “When contamination occurs there is rarely any way to confirm how it happened.” The argument that contamination must be proved suggests an analogy: that doctors should demand that the patient prove the exact moment at which he or she became infected before the doctor commences treatment.
In his 2010 report on the Farah Jama case, Former Australian Supreme Court Judge Frank Vincent wrote (p. 24), “Precisely how it [contamination] may have happened cannot be determined as the deposition of the minute quantity of material involved could have occurred in a number of ways. It is possible to speculate about the probability of transference through various mechanisms, but ultimately pointless to do so.” Judge Vincent also wrote (p. 45), “Whilst there is no absolute bar to conviction based solely on DNA evidence, the better view is that a conviction should only be returned where there is DNA evidence and at least one other item of evidence present which is consistent with the guilt of the offender.” The tiny amounts of DNA involved and the ease with which DNA transfers make it very difficult to pinpoint exactly when contamination occurs. Attorney General Rob Hulls said, “Mr Jama’s case, I think, highlights the need for everyone in the criminal justice system to better understand the nature and the limitations of DNA evidence. The perceived value of DNA evidence means that extreme care must be taken at every stage of the process: from collection of DNA to the handling of DNA, the testing of the materials and indeed, the interpretation of the results, to the way in which the evidence is presented to juries in criminal trials.”
Lack of good forensic technique
If the forensic police fail to follow generally accepted guidelines in collecting evidence, should the evidence be accepted? The CSC wrote, “Prof. Novelli had agreed that there are protocols and recommendations, but added that first of all the operator had to contribute his common sense (ud. 6.9.2011, p. Transcription 59.), otherwise it put in question all the DNA analysis done from 1986 onwards.” Does the CSC really believe that poor technique is insufficient ground for considering the evidence to be unreliable? The laws governing forensic evidence must set the correct incentives in any criminal justice system. If evidence collection procedures are flawed yet the evidence is accepted anyway, there is no impetus to collect it properly: The same types of errors may convict innocent persons in other cases. In the Busco case, Professor Novelli sounded a more cautious note than in the present one: “According to Professor Giuseppe Novelli, ordinarius of genetics at Tor Vergata, ‘There were contaminations among items and for [=of?] the analyses on the corsage and on the bra. The chain of custody of the items did not respect national and international standards for the conservation of items.’” What common sense actually suggests is that the standards were put into place to avoid generating a misleading result and that if one violates a particular protocol there should be a solid, clearly defined reason. The CSC provided none; on the contrary, its words are a smokescreen rather than an argument.
The CSC effectively rejected the idea that the onus is on the prosecution to show that the forensic police followed correct protocols. Not everyone agrees, at the very least. Based upon a number of cases, a barrister in Australia argued that the prosecution must demonstrate that it has done everything correctly in a preliminary hearing. Peter Faris wrote, “DNA should never be admitted unless there is corroboration: that is, there must be some other evidence of the identity of the offender. As for the rest of DNA cases, the solution is to put the burden on the prosecution.” Mr. Faris continued, “For example, the prosecution would be required to produce evidence to disprove contamination, whether at the scene or in the laboratory. Strict proof of continuity of exhibits would be required. The prosecution would have to call evidence of the current international statistical procedures. Proof by the defence of international articles and learned writings could be relaxed.” These are much more sensible guidelines than anything provided by the CSC.
Negative controls and the importance of electronic data files
Another serious objection to the court’s position is its belief that a witness for the prosecution need only testify that controls were run, as opposed to actually producing the negative control data in discovery. Negative controls are runs in which template DNA is deliberately left out. A good way to determine whether or not global contamination occurred is to examine the negative controls in the form of electronic data files, because if any DNA shows up, it must be from contamination. Among the reasons why examination of negative controls should be done using the electronic data files (EDFs), which are the raw data used to construct electropherograms is that this allows the scientist to zoom in on small peaks. Given the smallness of some of the peaks in the bra clasp profile and of all of the peaks in the knife profile, negative controls in the form of paper copies in which the y-scale were set to two thousand RFUs for example, would be almost useless. Even if one accepts the dubious notion that the defense bears the burden of proving contamination, then it is indefensible to prevent the defense from having access to any and all documentation that bears on this possibility, including but not limited to having the EDFs: It is also essential for the defense to examine the laboratory protocols, instrument logs, contamination logs and corrective action files. Professor William Thompson noted, “Under a guideline issued by the FBI’s DNA Advisory Board in 1998, forensic DNA laboratories are required to “follow procedures for corrective action whenever proficiency testing discrepancies and/or casework errors are detected” and “shall maintain documentation for the corrective action.” Obviously, the FBI’s rulings are not binding on laboratories in another country. However, good forensic science, like any science, doesn’t change as one crosses a border from one country into another. Good forensic principles, such as keeping a corrective action log, deserve to be adopted universally.
Given their importance in detecting contamination, it is not altogether surprising that negative controls themselves may be the subject of forensic fraud. Professor Thompson continued, “DNA analysts have recently been fired for scientific misconduct, and specifically for falsification of test results, by a number of forensic laboratories, including labs operated by the FBI, Orchid-Cellmark (another large private DNA laboratory), the Office of the Chief Medical Examiner in New York City, and the United States Army. In all of these cases, the analysts were caught faking the results of control samples designed to detect instances in which cross-contamination of DNA samples has occurred.” In the case of the fraud committed by Jaqueline Blake, Professor Thompson argued that if her superiors had examined the EDFs, it could have uncovered this misconduct.
The CSC made much of Professor Novelli’s testimony to the effect that he found no evidence of contamination, but what criteria he used were not provided. The CSC should state whether or not Professor Novelli used the electronic data files in his examination. If Dr. Novelli did, then he had evidence that has been denied to the defense, which raises the very troubling question of why the prosecution and its witnesses should have access to something that was denied to the defense and its expert witnesses. If he did not, then his review was incomplete, and it makes his claim that there was no contamination quite dubious at best. Every DNA expert whom I have consulted or whose thoughts on the matter I have read has been unequivocal in the importance of reviewing the EDFs; that they should be released in routine discovery is a key recurring subject in this blog. Dr. Mehul Anjaria wrote, "DNA analysts import ‘raw’ data from capillary electrophoresis instruments into software that assists in evaluating the DNA profiles. The DNA analyst can review peaks by zooming in and looking at height, morphology, and location to assist in determining if they should be reported as DNA alleles. The printed data can be edited by the analyst to show only the peaks being reported. Thus, it is imperative that a reviewer have access to the raw data and be able to independently evaluate the raw data in the necessary software to determine if the reported alleles are consistent with the actual data. The laboratory’s interpretation guidelines are another necessity for the review."
Independent case reviews (including but not limited to an examination of the EDFs) turn up problems in about two thirds of all cases, according to Professor Dan Krane. The Patrick Waring case in Australia is just one of many examples. Dr. Theodore Kessis commented on the Benjamin LaGuer case: “It is highly improbable that any given forensic DNA laboratory will take it upon itself to contact its accrediting bodies or the press and state for the record how often they make mistakes… To best understand the weaknesses associated with DNA testing we must rely upon the empirical, the occasions in which such deficiencies are revealed either by the press or internal review of a lab’s documentation of such problems by a defense expert. A close look at either reveals that indeed many instances of DNA testing errors have lead to the false conviction of individuals.”
Assertions from witnesses for the prosecution cannot be taken at face value
What should we make of Professor Novelli's claim, which the CSC accepted, of having examined hundreds of pieces of evidence and not observed evidence of contamination? This is problematic on several grounds. First, it is unclear what Professor Novelli’s criteria or methods were for proving or disproving contamination. Therefore, there is a decided lack of transparency in the process. Second, Professor Novelli did not explain the presence of multiple profiles on the clasp (see part B). Third, if the CSC accepts the judgment of a prosecution witness over independent experts, it suggests that the court has a strongly pro-prosecution bias. Nor can it be persuasively argued that Professor Novelli’s credentials in the area of DNA forensics are stronger than those of the independent experts, Conti and Vecchiotti. Professor Novelli’s main research interest is in the area of medical genetics with some additional research into one specific area of forensic genetics (that of single nucleotide polymorphisms), whereas Conti and Vecchiotti have focused more narrowly on forensic genetics.
Fourth, forensic witnesses make false statements while testifying. The CSC accepted the prosecution’s claim that negative controls were performed, despite tacitly acknowledging that they were not part of the case file. In his report on the Leiterman case, Dr. Theodore Kessis highlighted the fact that a negative control electropherogram showed contamination, and that Dr. Milligan labeled it as such. Despite this, Dr. Milligan later testified that no contamination occurred and that if it had, his reports would have documented it. In “Tarnish on the Gold Standard” Professor William C. Thompson wrote, “The DNA analysts in the Houston Police Crime lab came up with an easy solution— they simply failed to run extraction blanks (although they claimed in testimony that they had run all necessary controls).” Thus personnel from crime labs sometimes falsely testify that they followed protocols and saw no evidence of contamination. Furthermore, even if the lab itself detected no problems, independent case review often does (see above). The implied standard of the CSC (proof by assertion of the forensic police and the prosecution’s witnesses without documentation) flies in the face of the principle that justice must be seen to be done. It is negligent of the court to accept that when a prosecution witness asserts something as true, the assertion should be sufficient as proof that it is.
Ms. Stefanoni claimed that there had been no contamination in her lab in seven years, but it is difficult to see how a large lab could have an error rate of zero. Reporting on some crime labs in California, Maura Dolan and Jason Felch wrote, “The number [of errors] reported was small considering overall caseload -- 3,100 over five years – but [UC Irvine Professor William C.] Thompson said mistakes caught by labs ‘undoubtedly’ make up a small fraction of errors. (In fact, he said, labs that report the most are probably better run than those that claim none.)”
The Six-day gap in testing
A central pillar of the CSC’s report (p. 68) is that the six-day gap in testing the knife was sufficient to rule out laboratory contamination: “Dr Stefanoni (technical consultant who wrote the advice art. 360 codaproc.pen.), heard also on appeal, had repeated that there was no evidence of contamination: investigations on the knife had been conducted six days before the last DNA trace of the victim, then the analysis had been blocked for a further six days, a period deemed by the same expert Vecchiotti time to be sufficient to prevent laboratory contamination, as declared in the SAL report, wrongly reported as missing initially.” Whether this assertion is true is difficult to verify without full discovery taking place. It is not clear whether all of the DNA electropherograms were released to the defense, and what might be on the possibly unreleased electropherograms obviously cannot be known. For the sake of argument, however, we will assume that there was a six-day gap. The CSC cherry picked one statement to bolster its preferred conclusion; both Conti and Vecchiotti still believe that the DNA evidence is unreliable in spite of the gap. Moreover, the six-day argument does not address the possibility of secondary/tertiary transfer before or during collection of the knife at all (see part B below).
The assertion that a six-day gap is sufficient to exclude laboratory contamination is flawed on at least four grounds. One, no primary or secondary source in the forensic literature has made this claim, to the best of my knowledge. Two, any rule of thumb to that effect that was generated with respect to ordinary amounts of DNA may or may not be valid with respect to low template amounts of DNA. In response to a question of mine Dan Krane replied, “Was Meredith's DNA processed in the lab before the knife? If so, then I don't accept that six days is sufficient to exclude contamination in the lab. That would just be bad practice, plain and simple.” Meredith’s reference profile was indeed generated before the six-day gap and was obviously much higher in amount that the knife profile, which falls into the low template range. This is doubly problematic. Generally, one wants to run the reference profile last, because it is in high quantity, but low template quantities of DNA are especially worrisome. In response to a previous question of mine Dan Krane wrote, “There is absolutely no question but that contamination is a much greater problem in LCN cases than conventional DNA testing. The reasons that it is a greater problem are both because it is easier to detect contaminants ([Sara] Gino's point) and because it is easier to transfer (and to transfer without knowing) smaller amounts of DNA than larger amounts of DNA.”
Three, imagine that a forensic worker uses a tool such as a ruler in his or her examination of one piece of evidence, transferring DNA to it. Then the ruler is not used again for a week. If it were not properly cleaned, the ruler would be quite capable of transferring DNA to a new item of evidence. A recent study of the surfaces and instruments used in autopsies indicates that concerns about contaminated surfaces are well-founded. The authors wrote, “Using DNA-free swabs, we took samples from instruments used during autopsy and autopsy tables. Surfaces and instruments were routinely cleaned before sampling. Swabs were subjected to different PCRs to quantify the total amount of DNA and to amplify individual specific STR-markers. In most samples, alleles that could be linked to bodies that had been autopsied before were found. Furthermore, we could show that a DNA transfer from the autopsy table to a body was detectable in four out of six cases investigated.” A review article on trace DNA cautioned, “Fingerprint brushes are able to transfer amounts of DNA between exhibits that could generate profiles and may retain biological evidence for a considerable period of time [205,206].” Dick Warrington also warned of the dangers of cross-contamination from instruments. In his report on the Gary Leiterman conviction, Dr. Theodore Kessis wrote (p. 9), “It must be noted however that contamination errors have been documented where no direct processing link between sample and contaminant have been established, raising the specter that a source of contamination can linger in a laboratory for some time.”
Four, in the Jaidyn Leskie case there was a two-day gap in between examination of the condom used in the alleged rape of Ms. P and of Jaidyn Leskie’s clothing at the laboratory. The police ruled out the possibility that Ms. P was responsible for Jaidyn’s death, and Professor Krane’s examination of the electronic data files suggested that the possibility of a coincidental match was quite unlikely. Therefore, the only plausible explanation is contamination at the stage of handling evidence. There was also a one-day gap in the examination of two women in the Farah Jama case, and these two examinations are likely to be the cause of the observed DNA contamination, as discussed in the Vincent Report. Given that one-day and two-day gaps are demonstrably insufficient to ensure a lack of contamination, then why would a six-day gap be sufficient?
Observation of the collection or testing of the evidence
In the passage from p. 69 (“documented reporting activities carried out under the eyes of the consultants that had nothing to detect…”) quoted above the court seems to put emphasis on the observation of the collection and seemingly also the laboratory work by the consultants employed by the defense. This raises some serious questions. With reference to the collection techniques of the forensic police, the court’s stance is nonsensical. On the one hand, the court notes that the consultants did not object. Yet the court also wrote, “the vehicle of contamination must be identified in order to defuse the data offered by the technical consultants, it not being enough to assume insufficient professionalism of the operators in sampling…” (pp. 68-69) The court implies that pointing out technical flaws using recordings of the evidence collection long after the fact would not be good enough, despite the fact that this is what consultants or independent experts who are brought into the case later on would have to do. Equally serious is the fact that no one can see DNA. Therefore, when one observes the collection of the evidence, all one can comment on is whether or not the police used good technique (a great deal turns on the meaning of the word assumed). It seems as if poor technique is not sufficient in the court’s eyes unless it somehow demonstrates the vehicle of contamination, which it is quite unlikely to be able to do. On the one hand, the best technique does not guarantee that contamination will not happen; on the other hand, errors in technique increase the chances that it will.
If the court also means say that one must raise objections during the observation of testing the items of evidence, then their stance is equally problematic. Dan Krane observed, “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.”
Sporadic and global contamination
The CSC wrote, “he [Novelli] excluded absolutely persuasively that the contaminant could be present intermittently and that DNA could remain suspended, and then fall on a particular item.” Despite what the CSC believes, contamination may be sporadic (“intermittent”), as well as global. Dr. Donald Riley wrote, “Negative controls also can't rule out contamination of individual samples. The individual samples lack individual signs of contamination if it occurs.” Other authors whose works are quoted elsewhere in this entry also made the point that contamination can be sporadic.
Conclusions, Part A
The CSC’s report suggests an almost willful misunderstanding of DNA profiling. Its failure to put any significant burden on the prosecution and forensic police is out of step with other nations. The CSC’s stance that contamination must be proved fails to recognize that the exact moment of contamination may never be established even after thorough studies, as were conducted in both the Jaidyn Leskie and Farah Jama cases. A claim that a six-day gap in testing is itself assurance of a lack of contamination is unsupported in the literature and the time gaps in those two cases (during which contamination must have taken place) undercut their contention. Even if one accepted the dubious argument that contamination must be proved, then it is incumbent upon the prosecution to turn over the electronic data files (including negative controls), machine logs, standard operating procedures, and other relevant forensic data. Perhaps the most serious error the court made is to accept the assertion that there was no contamination without the methods or standards of proof even being defined. Under this court’s implicit rules, the prosecution would need only to offer DNA evidence and perhaps testimony from a friendly witness about the lack of contamination to ensure that the DNA evidence would be accepted in any criminal trial. Even without such testimony, the lack of insistence upon unfettered disclosure would severely hinder the defense from ever successfully challenging forensic DNA evidence on the grounds of contamination or misinterpretation of the data. Under such rules false convictions based upon faulty DNA evidence are inevitable because a defense based upon a theory of contamination would be virtually impossible to prove.
Part B: Specifics of this case
The CSC ignored innocent DNA transfer to the knife as an explanation for the knife DNA profile. It is possible that Meredith’s DNA was deposited onto the knife before or during collection by secondary or tertiary transfer, as was suggested by Dr. Alexander Kekule, among others. Professor Gregory Hampikian’s study of DNA transfer via gloves is further evidence of the plausibility of such mechanisms. Dried blood is a good source of DNA because small particles can flake off easily, and clothing is a known carrier of DNA. One can envision tertiary transfer from airborne DNA to officer Gubbiotti’s clothing, to his gloves to the knife. However, it is not up to the defense to prove a particular route of contamination; instead, it is up to the prosecution to show that they did everything correctly, as discussed in part A. There was no reason whatsoever for anyone to unpack and repackage the knife. Compounding the error, it was unwise to assign officer Gubbiotti to anything having to do with evidence collected at Sollecito’s flat, given the fact that he had been to the women’s flat on the same day. Reporting on the Patrick Waring case Estelle Blackburn wrote, “In court, police conceded they had not followed best practice in the case. Various officers said that the Central Park scene was left unguarded from 1.25am on the night, it was a week before it was searched, and the same officers had visited the homes of the girl and the accused which allowed for contamination of evidence.” What is not best practice in Perth is not best practice in Perugia.
The CSC's report does not seriously consider the lack of blood on the kitchen knife the prosecution has argued was one of the murder weapons. Yet the lack of blood plus the presence of starch call into grave question whether the DNA seen in the profile was actually physically on the knife. It is doubtful that one can clean a knife of blood and not of DNA, whether the scratch on the knife observed by Stefononi were real or not. Detergent water should lyse human cells, releasing DNA and other cellular contents. Bleach is used routinely to destroy unwanted DNA in molecular biology laboratories. If the knife were cleaned with bleach, the remaining DNA might show evidence of degradation (peaks associated with long DNA fragments would be smaller than those associated with short fragments). Yet there is no obvious trend of smaller peaks moving from left to right in the knife electropherogram. Moreover, there is no evidence that the knife was carefully cleaned; on the contrary, the presence of starch suggests that cleaning was not particularly assiduous. Forensic scientists Elizabeth Johnson and Gregory Hampikian wrote, “it is unlikely that all chemically detectable traces of blood could be removed while retaining sufficient cells to produce a DNA profile consistent with the victim.”
Another fundamental failure of the report stems from the fact that the knife profile is a low template sample. The CSC wrote,
“The discourse of justification, as maintained by the plaintiffs, did not take account of the authoritative voices of dissent concerning the presence of contaminating agents; adequate explanation was not offered as to how this assumption had to cover only some (the most demanding in terms of defense) examined tracks and not others; but above all it is based on the
erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods
tested, the results of which could certainly be called into question, but for their probative value, not for the operations carried out by preceding contradictory technique, from which did not emerge critical profiles at the time, but only in retrospect (about the decision at First Instance had dwelt from p. 289 to p. 298 on an abundance of topics only partially refuted in an appropriate manner, so that equally significant were the observations of Dr Stefanoni, brought to the attention of the court of Second Instance, at the hearing on 6.9.2011).”
The implicit assertion that the Rome lab was clean enough for low template DNA forensic typing is made dubious by the fact that dedicated facilities have been constructed to carry out this sort of work. To combat the increased risk of contamination in low template DNA profiling, special facilities and handling techniques are needed, as noted in the New Zealand Herald: “The ESR has spent $1 million building special anti-contamination areas at its premises in Auckland, Wellington and Christchurch. Protocols are being developed for crime scenes where the LCN technique is used and for the handling of samples from collection through to courtroom. LCN crime scenes will be divided into cold, warm and hot zones hot being the crime zone. Clothes are put on and discarded at each zone to minimise the risk of contamination.” The crown prosecution service wrote, “The FSS LCN test requires an ultra-clean laboratory and so is more expensive and less widely offered than the standard test.... The site of this bespoke laboratory is remote from other DNA Units, operates stringent entry requirements, is fitted with positive air pressure and specialist lighting and chemical treatments to minimize DNA contamination.”
In the article “Setting Up a PCR Laboratory” Theodore E. Mifflin wrote, “Air handling. For extremely high-performance PCR laboratories that will be involved with detecting very-low-prevalence DNA or RNA molecules (e.g., infectious disease agents in clinical samples), additional measures may be necessary to prevent contamination from the air being recirculated between the pre- and post-PCR laboratories.” Dr. Mifflin’s main focus is pathology, but his points about low levels of DNA are germane to low template DNA forensics in that both situations use polymerase chain reaction (PCR) to amplify very small quantities of DNA. With respect to the knife profile it is especially worrisome that Ms. Stefanoni presumably used a Speed Vac to concentrate the DNA. The sample would have been vulnerable to airborne contamination upon release of the vacuum or to any DNA left in the Speed Vac from previous samples. At the very least, a control should have been run that was also subjected to the same concentration step.
Indeed, the need for specialized facilities and handling techniques when working in the low template region is recognized even by the prosecution’s own expert witness. A review article coauthored by Giardini, Spinella, and Novelli stated, “Thus in these conditions [less than 100 picograms of DNA] there is a greater probability of artefacts, partial profiles with fewer alleles, contamination, preferential allele amplification, the complete absence of one allele (allele drop-out) in heterozygous loci and the nonspecific generation of extra alleles (allele drop-in) [78,79]…. Very few laboratories perform low template DNA typing properly, because it requires dedicated facilities and great experience, although there are several published methods for the interpretation of such profiles [80-82].” At the time of the murder, the Rome lab was not even certified for standard DNA testing. If a sample is handled in a regular DNA facility, without the special precautions available in dedicated low template DNA facilities, the risk of contamination is unacceptably high. Even if all such precautions are followed, some prominent DNA scientists find LCN DNA unreliable for forensic profiling. Nothing in the CSC’s report can be construed even as recognition (let alone an honest assessment) of the special problems inherent in low template DNA forensics or the lack of special precautions taken in the Rome lab.
The CSC’s views on the bra clasp profile are at least equally problematic. This blog has previously discussed DNA contamination on several occasions. Van Oorshot and colleagues wrote, “From a theoretical perspective, any DNA deposit that is not immediately relevant to the crime being investigated can be viewed as contamination. In this light, gross or sporadic contamination may appear at any point: (1) before the crime has been committed; (2) in the interval between the crime and securing the crime scene; (3) during the investigation of the scene; and/or (4) within the laboratory.” There are alleles in the autosomal DNA profile that are unidentified (not belonging to Meredith Kercher, Raffaele Sollecito, or Amanda Knox). There are also extra alleles in the YSTR profile that do not belong to Raffaele Sollecito. Even using a conservative threshold of 50 RFU, one locus has DNA from two additional males. If one uses a threshold closer to that employed in the knife electropherogram, the number of loci with two additional alleles (therefore two additional donors) goes up. It is unrealistic to posit that all of these alleles were deposited during the crime, leading to the conclusion that it was deposited in a way that is unrelated to the crime. Therefore, the bra clasp has already been shown to be contaminated by the Van Oorshot definition.
The CSC noted that a cigarette butt was the only other piece of evidence where Sollecito’s DNA was found (p. 68). They go on to make the fallacious and foolish equivalence that found DNA equals all DNA. In other words the court believes that the cigarette is the only source of Sollecito’s DNA in the women’s flat. Yet Mr. Sollecito left fingerprints and presumably used a towel to wash his hands before preparing food. Unless the forensic police sampled everywhere in the flat, there is no reason to believe that they found all of Mr. Sollecito’s DNA.
They also discount the problem of the six-week gap between the crime and the collection of the clasp, saying that the house was “boarded up.” This is highly misleading: the police themselves moved Meredith’s mattress and other items from one place to another. The bra clasp itself moved more than a meter from the point at which it was first seen to the point at which it was collected. The reason that the forensic police should do their work first is to collect samples before other police officers potentially contaminate the scene. The American Bar Association’s standards for collecting evidence reads in part, “Standard 2.1 Collecting DNA evidence from a crime scene or other location
(a) Whenever a serious crime appears to have been committed and there is reason to believe that DNA evidence relevant to the crime may be present at the crime scene or other location, that evidence should be collected promptly.”
The reason for being prompt is to minimize the chances of contamination: “The most important aspect of evidence collection and preservation is protecting the crime scene from the time the first officer or responder arrives until the last piece of evidence has been noted and collected without being contaminated.”
Furthermore, the bra clasp was allowed to decompose during the time that the forensic police stored it. If it could be retested, it is possible that additional evidence of contamination would surface. The CSC appears oblivious to this additional handicap that the defense faces, due to the impossibility of retesting. Some commenters have argued that when a piece of evidence goes missing or decomposes while in police care, the court should be obliged to accept the defense’s interpretation of the evidence. That principle would produce a more just state of affairs than exists presently.
The CSC wrote (p. 69), “…in a context in which negative controls were made by Dr. Stefanoni, checks that had been stated too superficially to be missing by the experts, simply because they were not attached to the report.” This is a tacit admission that the negative controls were never turned over to the defense. An anonymous expert in Italian law (one who is unaffiliated with this case) told me that an Italian lawyer can get anything he wants in discovery. Yet the electronic data files were never released to the defense during the trial of first instance, nor is there any evidence that they have been released since. Perhaps negative controls were simply not run (this would not be the first time in the history of DNA profiling that controls were simply left out). Even if one claims that the negative controls were made available in some format, it is certain that they were not made available in the form of EDFs.
Moreover, the lack of forensic discovery was more pervasive than solely the failure to release the EDFs. In an article from 25 April 2010 Fiona Ness quoted Greg Hampikian “’It’s fairly routine in the US that I send a request and get what I want. But in the Knox case I haven’t been able to get a copy of the standard operating procedures of the lab and without that, it’s hard to see if they even followed their own guidelines.’” Not only the EDFs, but also the standard operating procedures and machine logs should have been released but were not. In addition it is possible that a number of electropherograms were also withheld, some from the same time as the knife electropherogram.
The CSC was silent about the multiple failures of the prosecution to turn over forensic data and about Ms. Stefanoni’s false testimony about the quantity of DNA on the knife. Coupled with its fecklessness over Stefanoni’s stonewalling with respect to the results with tetramethylbenzidine, the court’s position contradicts any claim to the effect that an Italian lawyer can get what he wants. The CSC should have insisted upon complete release of all forensic information as a matter of principle, but even more so given the problematic, questionable nature of the DNA and presumptive blood evidence in this case. Moreover, to demand that the defense prove contamination without simultaneously demanding complete release of all forensic data, especially the negative controls, is absurd: Lack of complete discovery has repeatedly undermined the defense in this series of trials; therefore, the trials have been grossly unfair. The Duke lacrosse case might have played out very differently if North Carolina did not have an open discovery law, even one that is not perfect. Instead of correcting the failures to comply with reasonable discovery requests in the present case, the CSC has put its stamp of approval on them. If I were an Italian citizen, I would be asking myself whether the CSC would uphold the right to discovery more generally or would it be similarly obsequious to the prosecution.
The CSC wrote, “So the objective data collected indicating the absence of evidence (already highlighted in the judgment of first instance from p. 281 onwards, which made reference to the video recording of transactions that took place with the precautions of reporting protocols of the forensic team, accustomed to interventions of this nature) giving credit to the hypothesis of contamination…” They also wrote, “but above all it is based on the erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods tested…” (p. 69) Yet one of the defense consultants, Professor Potenza, did raise objections to the testing. Therefore, if the court is asserting that no one objected at this stage of the process, it is simply mistaken.
The CSC believes that the evidence collection was handled properly; this is one of the most remarkable and troubling positions it took in the section of the motivations document on the genetic investigations. For example, how could the CSC know whether or not the lab was clean? The independent experts, Conti and Vecchiotti, listed some of the recommendations concerning forensic evidence. “handling of the objects must be reduced to the minimum possible, and the objects must not be reopened [once packaged], not even for interrogation purposes..” and “Once sealed, the containers must not be reopened outside of the laboratory environment.” This guideline was ignored for the knife taken from Sollecito’s flat, as discussed above. Conti and Vecchiotti also quote guidelines concerning gloves that will come as no surprise to readers of an entry here, whereas Stefanoni’s beliefs on how frequently gloves are well outside the mainstream. Alex Heigl wrote, “There was laughter in the courtroom at one point when the jury was shown a video of the detectives collecting DNA evidence, doing the opposite of what the experts [Conti and Vecchiotti] had just described as the correct way.” Douglas Bremner wrote, “Professor Conti showed a film of the evidence collection that they said they had analyzed frame by frame in Amanda Knox case. ‘There are a number of circumstances that don’t follow protocol or proper procedure’ and the work of the scientific police was not supportable.” Does the CSC really believe that these errors never happened, or do they just expect a casual observer of this case to take their word for it?
"Everything is possible"
The CSC wrote, “The Court of Second Instance, supported the probable contamination advanced by experts, based on the "anything is possible", which is not an expendable [usable] argument, because of its generality…” The CSC plucked this phrase spoken under needlessly hostile cross examination and quoted it without context; it is a dismaying and troubling misrepresentation of Conti and Vecchiotti’s position. The Conti-Vecchiotti report used a variety of sources to document the manifold failures of the forensic team (see above). What they did not do, and what no one should have expected them to do, was to find a particular moment where contamination must have occurred.
The CSI effect and the cases of Farah Jama and Lukis Anderson
DNA evidence is based upon solid science, but there are some limitations inherent in DNA profiling that deserve due consideration from the justice system. Despite these limitations DNA evidence can exert an effect on the minds of judges and jurors that is out of proportion to the weight it deserves. This blog has previously covered the case of Farah Jama on several occasions, but the recent case of Lukis Anderson also underscores the problem of unintended DNA transfer leading to false imprisonment. A spokesman for the ambulance associated with the paramedics that picked up Mr. Anderson said, “We don’t have any definitive answers as to what may have caused the transmission of DNA going from one person to another.” The Anderson case is also additional evidence that one cannot easily determine a route of DNA transmission, even when it is obvious that it happened.
Conclusions, part B
The CSC accepted a number of extremely dubious arguments with respect to the DNA evidence in the Knox/Sollecito case while at the same time failing to address good ones. The CSC did not answer the question of how there could be DNA on a knife without blood. This paradox alone is strong evidence for contamination. Remarkably the CSC did not acknowledge the obvious and independently verified failures of the forensic police to follow good practice, both with respect to the knife and the bra clasp. Its position with respect to the bra clasp, that there was no way for one particular sample of Raffaele’s DNA to have made its way to the clasp, is at best terribly misguided. The CSC also did not acknowledge that the multiple profiles on the bra clasp force one either to claim many unknown assailants or to acknowledge that it is contaminated. On top of these other questionable positions, by failing to compel the release of the forensic data, the CSC eviscerated Ms. Knox and Mr. Sollecito’s right to challenge the evidence against them. The court’s stance not only puts Ms. Knox and Mr. Sollecito at risk of a false conviction, but doing so also greatly increases the odds that future defendants will be convicted on the basis of DNA evidence that is equally erroneous. One might hope that a nation’s supreme court would be a bastion against the state’s encroachment on an individual’s liberty, but the CSC’s report gave no evidence that such hope is justified; instead, the court has acted as if it has fallen prey to the CSI effect.
Former Australian Supreme Court Justice Vincent summarized the problem perfectly in his report on the Farah Jama case (p. 11). “In other words, the DNA evidence was, like Ozymandias' broken statue in the poem by Shelley, found isolated in a vast desert. And like the inscription on the statue's pedestal, everything around it belied the truth of its assertion. The statue, of course, would be seen by any reasonably perceptive observer, and viewed in its surroundings, as a shattered monument to an arrogance that now mocked itself. By contrast, The DNA evidence appears to have been viewed as possessing an almost mystical infallibility that enabled its surroundings to be disregarded. The outcome was, in the circumstances, patently absurd.”