Thursday, September 19, 2013

The Italian supreme court’s dangerously erroneous views on forensic DNA contamination

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.[19]”  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,[14] Orchid-Cellmark (another large private DNA laboratory),[15] the Office of the Chief Medical Examiner in New York City,[16] and the United States Army.[17]  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
Kitchen knife
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.

bra clasp
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.

Forensic technique
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.”


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David Anderson said...

Thanks Christopher for a very detailed and perceptive analysis of the CSC's incredibly biased 'analysis' of DNA evidence in this case. I shall send the link to Francesco Sollecito.

Paul Smyth said...

Excellent work. I will be rereading it I am sure. It is painful to realize that you are making a much more serious and rigorous examination of this issue than the ISC did. The high court has ruled on elements of this case three times now and in each instance their published opinion was intellectually lazy and quite embarrassing to Italy. The role of courts, it seems, is not to analyze and evaluate but to confirm the prosecution theories, no matter how bizarre and inaccurate.

Unknown said...

Excellent analysis Chris! I've read through the latest motivation several times & its mind boggling how the Group attack theory can even be considered. The evidence points to one man, Rudy. Their logic is absurd & this is an embarrassment to Italy.

Rose Montague said...

Great job Chris. You have stated tactfully and completely the problems with the ISC ruling on the DNA evidence. I will be a little more blunt. Novelli is a whore, paid to say what the prosecution wanted him to say even if it contradicted what he has said about DNA in the past. I also agree with Paul on his comment above. The ISC failed to do their job, they violated their own laws regarding making rulings and judgements on the evidence in this case and they appear to have read only the prosecution arguments and rubber stamped them. They are corrupt.

TomZ53 said...


Excellent analysis; comprehensive and well documented. You effectively articulate the illogic and inherent biases of the CSC report. An edited version reduced to explain the key points to a non-technical audience would be most useful in informing journalist, bloggees, and the general public about the overwhelming flaws in the prosecution's case.

Furthermore, in an academic setting it could serve well as an assigned reading in forensic science classes in a discussion of DNA contamination and ethics.

Chris Halkides said...

Rose Montague,

There is a long list of questions I would like to pose to Dr. Novelli. One of them is, "Can you find an example of a case of DNA contamination that conforms to what you and the CSC have said is necessary for proof?"

Gallagher said...


Let me assure you that the average Italian citizen does not go to his bed preoccupied with the Supreme Court or whether they are obsequious towards prosecutors. Rather, if he follows this case, he may well wonder, with some justifiable indignation, why taxes from his hard earned money are squandered on buffoons who squabble and are seemingly incapable of reading a simple DNA profile.

Your piece deals with contamination at some length and it's much appreciated, but if you can forgive an old English idiom, perhaps you can't see the wood for the trees. In other words,before analysing and contending the various contamination issues, shouldn't we first address the fundamental problem here, which the Supreme Court has highlighted - that is the complete disparity of so called expert opinion with interpreting the DNA profiles.

Novelli was adamant regarding the alleles from the clasp, a chance of I in 3 billion. Toricelli claimed the Y haplotype was so clear in all 17 loci that the database only yielded Sollecito. Stefanoni obviously agreed with this, as did other experts. Now, how is this to be reconciled with the views of Conti , Vechiotti and other defence experts and consultants. Hellmann states there was an erroneous interpretation of the electrophoretic profiles. Just how many ways can an expert interpret a DNA profile? Does it depend on his qualifications, or does it depend on who is paying him? Who is right and who is wrong here, and what does it take to have a profile read? Wouldn't you think that someone,somewhere, could take care of this. I have a vague suspicion that whoever solves this conundrum will also resolve the great mystery of contamination.

Chris Halkides said...


Nice to have you back. Conti and Vecchiotti do not dispute that Sollecito’s profile is present, and neither do I. However Vecchiotti and Zoppis coauthored a recent article about the case that was reviewed by Balding and one other individual. It makes some of the problems in Stefanoni’s work a little bit clearer. The authors wrote, “The DNA extracted from the bra clasp thus indicates the presence of several minor contributors, which was not disclosed by the scientific police…The genetic profile thus derived from a mixture of unidentified biological substances, whose larger component corresponded to the profile of the victim and whose smaller components suggest the contribution of several male sources.” If one claims that these alleles come from “the environment” (whatever that is supposed to mean), then one is in effect acknowledging that the clasp is contaminated.

Vecchiotti and Zoppis are on completely safe ground to criticize Stefanoni for classifying peaks as stutter which do not fall into the stutter position (one repeat-unit shorter than the main peak). They also noted something about the bra clasp about which I was unaware: “It could not be analyzed by the Appellate Court experts as it had been stored by the scientific police in a tube containing extraction buffer, which made it completely rusty.” If one stores an item in a closed container with water present, no wonder it decays.

One of the prosecution’s expert witnesses gave testimony that appears to be inconsistent with what he has previously said or written. That the court would believe the prosecution’s witnesses over the independent witnesses is very troubling. More generally the interpretation of mixtures is known to have an element of subjectivity, partly through the work of Itiel Dror and Greg Hampikian (link to The Economist article that summarizes their work here). Vecchiotti and Zoppis indicate that they are well aware of the problem of a suspect-centered analysis: “First of all, interpretation of a profile obtained for a particular item that is deciding which electrophoretic peaks are allelic and which are stutter or other artifact, must be done without reference to the suspect's profile: it is the only way to minimize the risks of bias in the interpretation of the profile derived from the evidentiary sample.” I wish I could convince myself that Stefanoni followed this important rule.

Pitchforks said...

This is a very cogent and readable scientific analysis of the appallingly flawed and blatantly biased Corte Suprema di Cassazione report on the conducting of DNA analysis “giving its reasons for overturning Amanda Knox and Raffaele Sollecito's successful 2011 appeal and sending the case for another trial in Florence.” It is also revealing of certain Italian cultural tendencies I outlined in my article The kind of intellectual dishonesty and illogical arguments exposed are alarmingly prevalent in Italy, and taint all aspects of daily life, never mind the personal ambitions of those in authoritative positions, politics and the criminal justice system. The incidental – and to outsiders, surprising - conceding of certain facts that belie the credibility of what is being stated is another typical aspect of Italian culture. Fiddling the system and corruption are regarded by Italians in general as a “given”, so that blatant disregard of what might be considered expected ethical practices in other nations is an unfortunate norm – everyone knows so why try to actively hide it, wink wink….? The banal analogy, and one that pretty much sums up this cultural attitude, is the knowledge by a whole community, and even his wife, that a man is having an affair, but in order to maintain the status quo and not disturb comfortable routines, traditional family structures and facades everyone turns a blind eye, including the betrayed herself. Italians are not ones to make huge waves, those running the country know this, so that outsiders often marvel at the brazenness with which unscrupulous activities are carried on with only perfunctory measures to cover them up. Hopefully the individual Italian citizens chosen as jurors will be able to step out of their apathetic cultural conditioning for the purposes of this trial and return a verdict that acknowledges the outrageous incorrigibility of the prosecution and of the biased agenda of the CSC.

Anonymous said...

A masterpiece, Chris! I have read it three times now.

The ISC did not do any DNA homework, doesn't express any interest in or seem to see any reason for doing any DNA homework and bases its views on (1) what seems to be complete ignorance about the relevance of DNA testing protocols and procedures and (2) shocking ignorance about what even makes DNA a reliable source of information for the court to consider.

One hopes that something may have been Lost in Translation in the opinion.

I challenge anyone to find a piece of legal writing from ANY COURT that demonstrates a less rigorous exercise of collective intellect. And this is from the Supreme Court of a whole country--where one would expect to find the country's most brilliant and best trained minds.

One understands and expects even transparent political leanings in court opinions, but these biases are almost always accompanied by reasonably sound intellectual underpinnings. One really does not expect sheer stupidity.


Chris Halkides said...

Tools in a forensics lab can hold onto DNA for a long time. Van Oorshot and colleagues wrote, "The profile obtained from the magnification lamp was entered onto a database of laboratory generated DNA profiles and found to match a number of samples, all related to one case, including two evidentiary samples that were examined on the bench associated with the magnifying lamp in question. These two samples, one bloodstain and one trace, were taken from the same item, described as a 'bulky jacket,' 3 months prior to the swabbing of the magnifying lamp. The magnifying light would have been used during this examination (personal communication with the analyst). As it is unlikely that the exhibit came into direct contact with the top side of the lamp we suggest that the likely cause of this DNA appearing on the lamp is due to DNA-containing material transferring from the jacket on to a glove and then from the glove on to the lamp whilst examining the jacket." Three months is quite a bit longer than six days.

Anonymous said...

Arnt there quite a lot of convictions in the US that should be vacated on this basis? Convictions obtained purely on the basis of DNA evidence where there was no supporting evidence and where one could posit the possibility of contamination?

Chris Halkides said...

Anon., I can only think of one case in which someone is in prison despite the evidence for contamination being very strong. Gary Leiterman (who is hardly a perfect person) is probably wrongfully convicted of the murder of Jane MIxer. Perhaps not coincidentally, the state of Michigan is one of the very few jurisdictions to challenge the notion that the defense should have access to the electronic data files. There are probably U.S. cases in which DNA was the sole evidence, and if so, then I have some very serious reservations about whether the DNA evidence alone is enough to warrant a conviction on the BARD standard.

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jdcastro said...

As far as I am aware, the luminol footprints had to have been made in blood (the iron in the haemoglobin causing the glow). Consider that:

1. Some bleaches can catalyze the reaction, also causing the blue glow. However, in this case the tests were done over a month after the murder, by which time the catalyzing wouldn't occur due to the evaporation of the bleach

2. If the scene was cleaned with bleach, any luminol reaction to it would have showed up over the whole floor area, not just confined to the footprints.

Therefore there are two possibilities that flow from this: either (1) the scene was cleaned with bleach which subsequently all evaporated, or (2) the scene was cleaned with another substance such as water

In either case the only viable substance remaining to cause the reaction is blood.

Unless you believe that the perpetrators applied horseradish or turnip sauce to the soles of their feet, which in my opinion is extremely unlikely.

Of course, this is only one of a great many evidences against Knox and Sollecito, even if you exclude any DNA results.

Chris Halkides said...


The premise of your argument is in error. Many substances are luminol-positive, including rust, copper ions, and animal blood. Because of the well-known problems with false positive reactions, some jurisdictions do not allow positive luminol results into the courtroom unless it is backed up with additional evidence. Yet no true confirmatory test was ever run. Both the negative results with TMB and the lack of Meredith’s DNA argue against the prints being blood. In a previous blog entry I quoted two independent, internationally recognized forensic chemists who indicated that the footprints in the hall were probably not made in blood. In other words the additional evidence is either not present at all or the evidence pointed to the opposite conclusion that the luminol suggested.

In addition one commentator at the James Randi Educational Foundation discussion has said that one luminol print lies in the same place as a bloody shoeprint that the police wiped away. If correct, his interpretation suggests that the substance that is reacting with luminol is bonded to the floor tile in some way. With respect to cleaning and luminol there are usually still streaks from small amounts of hemoglobin left behind. Nor does the hallway look as if it had just been cleaned, although I have not spent a great deal of time looking at the photos.

There are additional problems with the luminol results. The luminol was overapplied, leading to dilation of the image and loss of detail, according to Colonel Garofano. Considering this problem and the lack of reference prints from all but three people, I cannot see how they could be attributed to any one person. In addition, the work was done more than six weeks after the crime, and after there had been a great deal of foot traffic in the flat. Amanda walked through there following morning, and others walked through the hall in the afternoon. There are photos of the forensic police stepping in Meredith’s dried blood. Therefore, even if the luminol positive prints were made in blood, there are innocent explanations for blood being tracked around. Finally, prints themselves are all right feet and do not form a trail. They don’t tell a coherent story.

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