To those who believe that Amanda Knox and Raffaele Sollecito are guilty, I issue a challenge.

Part 20 in the Knox/Sollecito case

Francesco Maresco, lawyer for the Kercher family, said after the verdict last December, “We are teaching the whole world and the United States how to collect, analyse and evaluate scientific technical aspects of the case.”

I have previously discussed how the electronic data files are used by independent experts in criminal cases. Those arguments are especially compelling in the murder of Meredith Kercher, in which the amount of DNA in two of the most important pieces of DNA evidence fall near or into the low copy number (LCN) range. The profile from the bra clasp is also a mixture, and it is well known that the analysis of mixtures introduces subjectivity into the analysis. I have also previously given my reasons for believing that these critical files were never released to the defense teams. Since that time some additional information has become available that puts this question to rest.

Raffaele Sollecito’s appeal documents indicate that his defense expert Dr. Pascali requested the raw data in 2008 (presumably the electronic data files). This request was prior to the preliminary hearing, and the lawyers supported their request with a reference to article 415. They also requested the log files. The judge granted the defense teams the right to forensic information in the summer of 2009 that they had been denied until that time. However, the prosecutions did not fully comply with this order, according to a source close to the defense, and this was one of the things that precipitated the defense teams’ asking for a mistrial in September of 2009.


An open letter coauthored by Dr. Elizabeth Johnson and Professor Gregory Hampikian noted that the prosecution failed to release the electronic data files (.fsa files) to the defense. Since the time of the letter’s release on 11/19/09, some have assumed that these two DNA forensic scientists were unrelated to the case and therefore had no standing to ask for these files. This argument is false. An article written by Fiona Ness on 25 April 2010 indicates that Professor Hampikian is a consultant for Amanda Knox’s defense team. As such, he has every reason to request the operating procedures of the lab, as well as the electronic data files that produce the electropherograms.

“The importance of DNA evidence in a conviction is highlighted in one of Hampikian’s cases - that of US student Amanda Knox. Knox was convicted of the murder of British student Meredith Kercher after a high-profile trial in Italy in 2009. Knox’s conviction was partly based on a sample of her DNA discovered on a knife found in the house. However, Hampikian says he is appalled by the standard of DNA evidence used by the Italian courts to convict Knox in the case. ‘The amount of DNA present is far below the normal cut-off for many labs that I know of. A one off spurious piece of DNA evidence shouldn’t be treated as a gold standard profile from a high-profile source. The issue of this low-template DNA sample is really troubling me.’ Trying to obtain the evidence to have it retested, however, is proving difficult. ‘The discovery rules in Italy are different to what I’m used to. 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.’ Evidence, Hampikian says, is a matter of science, to be reviewed, not protected.” (emphasis added) An article in the Irish Times confirms that a professional relationship exists between Professor Hampikian and the Knox family.

The words of an anonymous forensic DNA expert unconnected with the murder of Meredith Kercher are apropos here. This individual said with reference to a different case, “Honest differences of opinion by qualified experts should be welcomed by the Court. This can only be accomplished if the independent expert is provided full and complete discovery by the government.” Keith Inman and Norah Rudin gave ten ways to improve forensic science. Number five was “Provide transparency. Secrecy and gamesmanship are inappropriate to the work of the forensic scientist. All laboratory notes, data, results, procedures, logs, and records should be open to controlled
and appropriate scrutiny.”

To those who believe Ms. Knox and Mr. Sollecito are guilty, I respectfully issue a challenge: Join me in calling for the release of all of the forensic information that the defense has been denied until now. Some among you have argued that the Italian authorities should not release any more information than that demanded by Italian law. However, it is conceivable that Italian law is not yet settled on the question of whether or not to release the electronic data files. If it is not, why should the precedent be against full disclosure rather than in support of full disclosure? If the case against the defendants is as strong as you say it is, you have nothing to fear. On the contrary, your certainty would be stronger if the release and examination of these files produced nothing.

To the Italian authorities I respectfully issue a related challenge: Make good on Mr. Maresca’s claim. Show the world that forensic evidence is to be shared with the defense. At that point the analysis will be complete. Provide the electronic data files (.fsa files), and the instrument log files, and include the contamination log files if they exist. Then and only then will you indeed be showing the world the right way to treat forensic evidence.

DNA transfer in Strangulation

Part XIX in the Knox/Sollecito case

Update 1, 22 August 2010

I communicated with an anonymous forensic nurse about their domestic violence program. If an alleged victim complained of an attempted strangulation, complained of a partner’s grabbing their arm, or showed bruising or redness, they have been swabbing that area for DNA, for two years. Positive results in at least one case helped to convict someone. This information confirms the reasonableness of swabbing Meredith’s body in appropriate places, although the bruise on the nape of Meredith’s neck may have been the result of her being thrown against the wall (as some have theorized). We do not know whether swabbing the bruised areas was done or not.
____________________________


PM Guiliano Mignini’s reconstruction of the murder of Meredith Kercher was shown to the jury in the form of an animated video. In his speculation Amanda Knox grabbed Meredith Kercher by the throat and slammed her against a wall. The video superimposed actual shots of Meredith’s bruises with Amanda’s animated hand to imply that Amanda’s action produced the bruises. Later, Rudy Guede and Raffaele Sollecito held Meredith’s arms back, and Amanda stabs her (Barbie Nadeau, Angel Face, p. 160). In forensics professor Carlo Torre’s reconstruction of the crime, the single assailant grabbed Meredith by the throat and stabbed her. The area under Meredith’s chin and her nape were bruised; the former bruising was the result of the knife and the latter when the assailant put Meredith down.

Although Mignini’s theory of the crime suggests a much longer amount of contact between the assailants and victim than does Torre’s, both imply contact. None of Amanda Knox’s DNA was found in Meredith’s bedroom or on her body. Raffaele Sollecito’s DNA was found on a bra clasp (although the defense contests this piece of evidence) but not the bra itself. Rudy Guede’s DNA was found in several places, including on the sleeve of Meredith’s sweatshirt. Let us examine some forensic DNA studies to see if they shed any light on this tragedy.

The study by Wiegand and Kleiber gives a case study in which DNA evidence was collected 48 hours after a strangulation. They wrote, “Strangulation marks were clearly visible on the neck of the victim. Epithelial cells could be removed from the neck of the victim using separate cotton swabs for the left and the right side of the neck. Only the swab from the right side could be typed and included the pattern of the suspect (Fig. 3), a result which corresponded to the autopsy findings (the right neck side showed a higher intensity of bleeding in the muscles than the left side indicating a more intensive pressure against the right side). Altogether clear results could be obtained using four STRs (TH01, VWA, FGA, CD4) demonstrating the high utility and sensitivity of the method described.” These authors also conducted simulated strangulations, and they reported a success rate of better than 70%.

The simulated strangulation study in 2002 by Rutty used both SGMplus and LCN amplification. The simulated strangulation experiment was done with periods between the force and the sampling were 1, 5, 10, 15, 30, and 60 min, 2, 3, 4, 5, 6, 7, 8, 24, and 48 h and 3, 4, 5 and 10 days. When the author used SGMplus, he observed a full profile of the offender 7 out of 29 times, and always in the presence of the victim’s profile. When the author used LCN all 17 experiments yielded offender profiles, with the majority being partial profiles. Dr. Rutty wrote, “Of the test neck swabs, 19 yielded positive amplification results using SGMplus, 12 showed a victim-only profile and 7 a victim and offender profile with a full offender profile detectable up to 6 h after contact. When LCN was used (17 tests) all showed the offender to be present for all time periods i.e. up to 10 days. In the majority of cases it was a partial offender profile with the majority of the amplification result being a full victim profile.”

Dr. Rutty stated that “When considering the apparent time periods of DNA survival, passive transfer of the offender’s DNA onto the victim’s neck could also explain the presence of offender DNA several days after contact.” This study did not break down the results by time periods between simulated strangulation and DNA collection; therefore, it is difficult to draw firm conclusions based solely on the data presented.

A 2008 study by Graham and Rutty reexamined the question of DNA transfer to and from strangulation victims with an emphasis on innocent DNA transfers that might deposit DNA on the neck of a victim. In 24% of samples collected showed nonself DNA on the simulated victim from third party sources. The authors believe that such DNA transfers might confuse an investigation.

Finally, it might be helpful to return to the subject of primary and secondary DNA deposition. The study by Lowe and coworkers in 2002 looked at DNA transfer from a good DNA shedder to a poor DNA shedder to an object. By definition transfer was secondary from the good shedder, and transfer was primary from the poor shedder. When mixtures were observed, secondary transfer from the good shedder provided the major component, not primary transfer from the poor shedder. This study illustrates the dictum that one generally cannot infer the mechanism of how the DNA was deposited from the DNA itself. Along with the study by Graham and Rutty, this work suggests caution in the interpretation of nonself DNA on the body of the victim of strangulation. A similar caution should also be applied in the interpretation of DNA on the bra clasp.

In addition to the case study discussed by Wiegand and Kleiber, there are a small number of news reports of strangulations that mention DNA. One is from greater Detroit, and another is from Chicago. However, these articles do not specify the place on the body that was tested for DNA. In addition to the possibility that DNA was collected from the neck, it is possible that the victim’s fingernails contained the perpetrator’s skin cells or vice versa.

There are no reports that ILE found anyone’s DNA on Meredith’s bare wrists or her neck, and it is unclear whether or not the forensic police swabbed for DNA in these areas. It is also unclear whether some areas on the neck would have been free enough of Meredith’s blood to allow swabbing for the assailant’s DNA. Nevertheless it is difficult to see why the forensic police should not have swabbed the nape of Meredith’s neck or her wrists. If they did and found nothing, it would be strongly exculpatory, although it might fall short of proof of innocence due to uncertainties over collecting enough DNA or collecting it quickly enough after the murder. If the forensic police failed to swab these areas, it would suggest that they did not do as thorough a job as one would wish.

Bibliography
P. Wiegand and M. Kleiber, “DNA typing of epithelial cells after strangulation,” International Journal of Legal Medicine (1997) 110 :181–183. abstract

G. N. Rutty, “An investigation into the transference and survivability
of human DNA following simulated manual strangulation
with consideration of the problem of third party contamination,” International Journal of Legal Medicine (2002) 116 :170–173.
abstract

A. Lowe, C. Murray, J. Whitaker, G. Tully, P. Gill, “The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces.” Forensic Science International (2002) 129(1):25-34. abstract

E. A. M. Graham and G.N. Rutty, “Investigation into ‘normal’ background DNA on adult necks: implications for DNA profiling of manual strangulation victims.” Journal of Forensic Science, (2008) 53(5):1074-82. abstract

Raffaele Sollecito's Appeal

Part XVIII in the Knox/Sollecito case

Update, 7/12/10

Here is a better translation of a portion of Sollecito’s appeal dealing with the bra clasp, provided by an anonymous translator:

“But there is more. The lack of full discovery was also found during the course of the trial:
- on July 18 2009, during cross-examination of the technical consultant of Raffaele Sollecito’s defence, Prof. Adriano Tagliabracci, there was an astonishing coup de theatre, in which the prosecution formulated a question which generated the suspicion that the Public Minister was in possession of further laboratory data never made available to the defence. In particular, the data relative to the quantity of the extract used for genetic analysis of the biological material found on the bra clasp of the victim;
- confronted with the reaction of the defence, the office of the Public Minister had to admit that there existed documents compiled by the scientific police never deposited at the conclusion of the investigations (contravening in this way the provision of Article 415 bis c.p.p.), nor sent to the Gup with the request for trial (in violation of Article 416, paragraph 2, c.p.p.).
In other words, the office of the PM denied a complete discovery of the documents of the investigation, extending this breach in the rights of the defence into the trial!”

This passage indicates that the prosecution failed to provide the defense with information relating to the single most important piece of evidence against Raffaele Sollecito all through the pre-trial and well into the trial phase. What is unclear from the appeal document alone is whether or not all information was ultimately released. However, other documents indicate that the prosecution never released all of the information that the defense teams sought.
___________________________________________________

The following is Google-translated from Raffaele's appeal document (pp. 51-57) with some minor edits. Although it is hard to decipher the meaning in places, it gives a picture of the defense continually asking for information on the DNA testing all throughout the preliminary hearing and trial, and the prosecution continually refusing. I have previously written about the importance of the electronic data files to a proper defense and about the near universality of this disclosure. I am eager to hear the prosecution’s explanation of how their refusal to hand over log files and electronic data files can be anything other than a trampling upon the right of discovery:


“And there is no doubt that, in terms of 'prosecution, failure to discovery on one of the main aspects of the process has been a considerable advantage over the defense, since the case involved Raffaele Sollecito is building right on genetic testing. In fact, it is useful to make a historical reconstruction of the case at hand:
- Following the conclusion of preliminary investigations, the defense could review the technical report of forensic genetic investigations in the signature of Dr. Patrizia Stefanoni, to which were attached tables who reported only the number of alleles, without which they would specify any other information or scientific data (for clarity, we define each variant allele of a gene, the genotype of an individual gene has a relatively Kit alleles he is in possession);
- Given the extreme importance of this for proof on indication of Prof. Pascali expert defense Raffaele Reminder, on June 24, 2008 - pending the period of 20 days under Article. 415 bis -, was made an initial request to Prosecutor acquisition diagrams in electropherograms whereby the forensic results came in content Technical report on forensic genetics. In particular, this request is have specified the absolute necessity (in order of presentation defensive pleadings and petitions under Article. 415 bis cpp) to have the aforementioned documents available to the consultant of the could examine it in detail;
- However, against all evidence, the prosecutor dismissed the request was deemed inadmissible 'because the option in all.art.415 cpp a cover secured and only those acts ';
- To deal with a similar unjust refusal, the defense made on July 3, 2008, a further instance in which it sought to 'acquire forensic laboratories of the numerical values and RFU peaks on all findings, or alternatively to obtain police Scientific CDROM containing the raw data and peak RFU. It was asked, Moreover, in case it was not possible to obtain copies of these data, that the Prof. Pascali was allowed to go to the police service science for a spot inspection of the data and make it directly computerized copy;
- Even that request, however, was inexplicably rejected on 11 July 2008 with the anodyne statement that 'on the charts of Forensic there are already bars with generic value on fluorescence peaks.
The defense then returned to a preliminary hearing with a serious cognitive gap, due to lack of awareness of scientific data used by the police in their scientific investigations. As we shall see, this story had a further follow:
- Preliminary hearing of 16 September 2008, the defense reiterated request to acquire numerical values RFU and peaks at all findings or, alternatively, to acquire the CD-ROM containing the raw data RFU and peaks. The request filed at that meeting was based upon both the incompleteness detected in the diagrams attached to the report, and by the repeated requests made to the office of the C>P> to obtain the missing data;
- After this request, the Gup could not help but detect need 'for purposes of deciding' to understand 'fully (...) as procedure has been adopted for the analysis in question, and whether there further elaborations that constitute the necessary support conclusions reached. 'Consequently, the Gup has ordered the hearing in heard of Dr. Stefanoni calling 'to accompany their testimony with computer media containing data requested by the defense. "The same judge in that case was aware the extreme importance of data capture whereas the investigations living would receive 'the most important elements all.impianto accusatory;
- On September 25, 2008 in observance of the provision of Gup is was deposited by association. Zugarini on behalf of Dr. Stefanoni, the CD-Rom containing the requested data. In this regard like to highlight as the immediate acceptance of the instance in question proves unequivocally from the outset the importance of having the data available full scientific;
- Hearing on September 27, 2008, was then represented to the Gup need to obtain additional documentation because incomplete data, therefore, was asked to Court to acquire the service files (log files);
- However, the Judge, after seeking an opinion to Dr. Stefanoni has also rejected the application based on an uncritical adherence to its comments, coming to claim that "not be needed information contained in the logs (data nell.esperienza forensic are never identified, resulting standards approved by the parameters internationally and which appear to be in use in all Laboratories engaged in Forensic Genetics).
Such an established procedure is not, of course, acceptable under no point of view, since the unconditional adherence to observations of Dr. Stefanoni, has led to support the contention accusatory, without the technical-scientific comparison of an expert third and impartial.
And indeed, if it had consulted a person not bound by any constraint on the charge (ie a neutral), the parties would certainly have benefitted. And 'quite clear that to ask to review its person (Dr Stefanoni) which produced that result is, in fact, nonsense: can not say, claim of self-discipline science by the same technician who testified that result. Any specialist will of course be pushed, albeit inadvertently, to defend the validity of their work, even in front of a simple request for clarification, and justify - after all, understandably, by his point of view - that all data provided are sufficient.

Was evidence showed the need to provide all documentation scientific defense.
It 'important to note that log files are much scientific data relevant, if not indispensable, especially in situations (such as the case in point) where the peak height must be assessed together with other parameters (such as area) is also higher to determine the plausibility or, conversely, the unlikelihood attribution to a person's DNA. So it is true that during the preliminary hearing in court, the Dr. Stefanoni - at the request of the consultant that the Prosecution asked if it was important given the "peak area" – said "Normally not. "However, what is more surprising is that the very Dr. Stefanoni candidly admitted that it had no other experiences on (p. 68 transcription.'s preliminary hearing Oct. 4, 2009). The opinion of Dr. Stefanoni, in fact, emerged in all its fragility when the consultant has made the defense Reminder noted a recommendation of the International Society of Genetic Lawyers, which states that the method used to interpret traces of mixed DNA and consider l.area l.altezza of peaks "(p. 99 of those transcripts.)
The GUP at this point, he invited Dr. Stefanoni to be received by October 8, 'the data relating to the calculation of peak areas related to the finding 165 / B indicated by prof. Pascali in the third column, in his name, all.allegato referred to the report filed, and diagram on the second run on the sample elettroferografica 36B.
The epilogue of this long run-up occurred on October 8, 2008 with the filing by Dr. Stefanoni the CD-ROM containing the data dell.amplificato electrophoresis sample DNA called 165 / B (finding piece of cloth with hooks), and the CD-Rom containing: Sample data info for the two races dell.amplificato electrophoretic sample of DNA known as 36 / B (Finding knife) electropherograms of the second electrophoresis.
As can be seen from the picture just outlined, the defense did not have provision, at the conclusion of preliminary investigations, all acts of investigation as required by law, so much so that this uncertainty lasted at least for the preliminary hearing stage (and beyond, as you say).
For example, consider the simple analysis of the peak - technical data that was provided to the defense until after the hearing at the hearing Preliminary Dr. Stefanoni, damaging irreparably the rights of defense -revealed that the amount of DNA used for analysis was quite modest, which led the consultant to the defense clarified, that the interpretation given to the forensic graphics has minimally complied with the guidelines laid down by the International Society of Forensic Genetics.
But there is more. The lack of full discovery was also found in during the process:
- On July 18, 2009, during cross-examination technical consultant Defense Raffaele Sollecito, Professor Adriano Tagliabracci, there was an astonishing coup de théâtre, as the accusation made questions that have created the suspicion that the prosecutor had hold additional laboratory data ever made available to parties. It was, in particular, data on the amount of extract used for genetic analysis of biological material found on the hook of the bra of the victim;
- In front of the defense reaction, the Office of Public Prosecutor had to admit that there are acts performed by forensic ever deposited at the conclusion of the investigation (in contravention Thus the provision of art. 415 bis cpp) or sent to Gup with the request of trial (in violation of Article. 416, paragraph 2, cpp).
In other words, the Office of the PM or denied full discovery of documents investigation, pushing this lesion of the right of defense to the debate! We will now give an account of evaluations of the Court of Assizes of Perugia. The sentence I can refer about the motivations of the order of rejection "of All.audience 14/09/2009 defenses of the defendants objected l.avv n the interference with the right defense, arguing in this regard, which were verified the void. After hearing the other party seeking to disregard l.exception, this Court issued l.decree, which read out with which l.exception of nullity was rejected and was available to proceed further "(p.7. above).
Passing the order of 14 September 2009, the Assize Court noted as follows: 'respect of invalidity l.exception advanced and illustrated memory today filed for violation of the right of defense question exception is not admissible, l.activity sampling and comparison of traces biological object activity.exception the nature of prior technical investigation covered with the possibility dall.article 360 for parties to participate in and comment in this context is not apparent that there has been no lesion of the right of defense, for the nullity that would assessed in a case of a 415 dell.article knows nzione not provided by any specific provision or by a 415 Criminal Procedure Code that principle of mandatory nature of the void during preliminary dell.audience compare 1/10/2008 GUP measure included in documents filed today in support the plea of nullity that aspect has been addressed and its exception has not been repeated in terms of which all.article Code 491 Nell.audience of Criminal Procedure also before this Court on 18.7.2009 issue was not raised in terms of the void under the compare the transcripts of the hearing on 18.7.2009, with particular Refer to pages 102 et seq and 114 et seq therefore logical to conclude that l.aspect in all.exception now advances to integrate a nullity ta question But to sanction by the Supreme Court ruled on the matter for example compare Cassation Judgement second section 29573 of 2006 and Section Supreme number 21593 2009 its fifth exception was belatedly raised. As for the broken and also being the subject of advanced today than that penalty is justified in the presence of a omitted on filing all.entire investigations in this case l.omission h a covered part of the supporting documentation'”

DNA contamination and DNA cold hits

Part XVII in the Knox/Sollecito case

This post returns to the problem of forensic DNA contamination. It also addresses the problem of partial profiles and adventitious matches, especially with respect to cold cases. North Carolinians might want to take special note of the John Puckett and Raymond Easton cases, inasmuch as North Carolina may join other states in demanding DNA samples upon arrest. My prediction is that as the size of a DNA database grows, we will see a greater number of cold case convictions that turn out to be erroneous.

Murder of Jane Mixer
We have previously encountered the conviction of Gary Leiterman of the murder of Jane Mixer. The presence of both Mr. Leiterman’s DNA and that of John Ruelas. The prosecution was unable to come up with a reasonable explanation for Mr. Ruelas’ DNA; he was four years old at the time and lived in another city.

Dr. Theodore Kessis reexamined the DNA forensics of this case:
“Review of the electropherograms associated with this negative control sample (NEG 041902) reveals that it was contaminated, a fact that cannot be disputed since Dr. Milligan himself labeled it with a note indicating as much (Appendix 8 - Electropherogram sample NEG 041902).
Remarkably, Dr. Milligan stated in his 7/15/02 testimony that no contamination events had occurred during the course of his testing and that if any had, he would have documented them in his reports (pg. 141-21 and 142-4). Equally difficult to rectify here is the fact that when asked if he had ever committed an error, Dr. Milligan's replied he could never recall making one. (pg. 162, lines 1-5)”

While agreeing with others that most contamination events happen when two samples are handled closely in space and time, Dr. Kessis also offered a caution, “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.”

Dr. Kessis found another deficiency: “As previously mentioned, Corrective Action files and Error logs form an important part of a laboratory's quality control and assurance programs by centralizing the documentation of errors within the laboratory. Mandated by the DAB Standards (Appendix 2, section 14.1.1), such data allows laboratory management to quickly and efficiently identify and address systematic problems that affect the reliability of testing results.”

“Given the thousands of samples, hundreds of cases, and number of people handling casework within this laboratory, the lack of a centralize system to track errors and discrepancy indicates that the lab had no mechanism in place to identify and deal with systematic problems such as contamination events.”

There are several take-home messages for students of the Knox/Sollecito case here. Contamination by more than one individual’s DNA is possible. The laboratories in question will sometimes claim to be error-free, as did the Rome lab. In such an instance it would be helpful to check the laboratory’s corrective action files, which are more likely to be accurate than someone’s memory. Whether the Rome lab has such files is unclear. Finally, when DNA results force one to an absurd conclusion (as with the kitchen knife), contamination or misinterpretation should be suspected.

The case of Gregory Turner
Gregory Turner was indicted and jailed for a murder when the DNA from a murdered woman in Newfoundland was found on his wedding ring. The ring also contained DNA from one other contributor, thought to be his accomplice. However, the lawyer sought out the names of the technicians from the lab and determined that the technician working on his ring also worked on the victim’s fingernails. Moreover, the DNA from the second contributor to the ring turned out to be the profile of the technician herself. After many months, Mr. Turner was released from jail. This particular case of DNA contamination is an example of where both primary and secondary transfer of DNA occurred. As did the Mixer case, it shows that contamination can transfer the DNA from more than one person onto an item of evidence. This is worth bearing in mind given the multiple DNA contributors to the bra clasp in the Knox/Sollecito case.

Profile N
As was true in the Jaidyn Leskie case, the victim of one crime can become a suspect in other crimes on the basis of contaminated DNA evidence. Sometimes it takes a major foul-up to prod a laboratory into corrective action. In New Zealand, the Wellington police were investigating two murders, dubbed Rex and Pad, as well as N, the victim of an assault.

Michael Strutt reported, “One caught up in the testing was a mild mannered Christchurch man who had been assaulted outside his local, the Hagley Arms Hotel, on 23 April 1998. Police had taken blood in order to eliminate his DNA from samples taken from the scene of the assault and sent it to ESR for testing. The result of this test came to be dubbed 'Profile N' by the inquiries which were to follow… The Christchurch assault victim was subjected to 'extensive police inquiries' for more than three months and his financial records were seized. This established what he had claimed all along, that he had not left Christchurch around the time of the murders. In fact he hardly left Christchurch at all… Although they never discovered exactly how the mistakes had happened they did determine that the 'Rex' and 'Pad' samples had been accidentally contaminated with DNA from Profile N at an early stage of processing at ESR's Mt Albert laboratories. Extracts from them sent to other laboratories for testing also returned Profile N. Numerous recommendations for improving oversight, record keeping and even laboratory ventilation were made.”

“During the inquiry ESR [Environmental Science & Research Ltd] was asked to check the results of all testing done in July 1998 for any further instances of Profile N. Although they initially reported that none were found, later retests showed a partial profile consistent with N in another sample from 'Pad'. They also revealed 25 other 'unexplained' profiles in 14 of the 36 samples tested that month.”

A group investigating these cases asked a manager of the forensic unit of the ESR lab to comment, “The Wellington murders you refer to were not DNA failures. The problem was picked up and investigated. Five separate investigations failed to find scientific proof for the cause of the anomalies. No direct evidence of either accidental or deliberate contamination has come to light. Existing facilities and protocols were shown to meet independent quality audit requirements. The Ministerial Inquiry recognising the points above concluded that on the balance of probabilities the apparently anomalous results could be ascribed to accidental contamination of DNA extracts within the ESR laboratory. They made a number of recommendations which ESR has either implemented or is planning to implement. This sits comfortably with our continuous improvement management focus.”

The fact that the lab in question met the audit requirements is evidence that simply following accepted protocols does not guarantee that contamination will never happen. The lack of the determination of an exact mechanism of contamination is consistent with Dr. Kessis’s caution in the Mixer case above. People who argue that Amanda Knox and Raffaele Sollecito are guilty sometimes demand a mechanism of contamination; unfortunately, this cannot always be determined.

The first false cold hit case in the UK
Michael Strutt wrote, “A man [Raymond Easton] with advanced Parkinsons disease who could not drive an automobile or dress himself unaided was linked to a burglary which had occurred 200 miles from his home. In spite of protestations of innocence and alibi evidence police arrested him because the DNA profiles matched and 'so it had to be him'. It was several months before10-point DNA tests were done on samples from the suspect and the crime scene. The results exonerated him.

He gained his freedom and a brief note from the prosecutor saying that charges were being dropped because ‘there was not enough evidence to provide a realistic chance of conviction’. He still awaits an official apology. Or even an admission of error.”

The reason why Mr. Easton was initially suspected was an adventitious match, as opposed to contamination. This case shows that adventitious matches can happen especially for partial profiles. The first profile had only six loci, but the second profile had 10 loci. As with DNA contamination, when a cold hit match leads to an absurd result, the DNA evidence needs to be reconsidered.

The Sylvester murder, a cold hit combined with a partial profile
According to Dr. Donald Riley, “A partial profile is one in which not all of the loci targeted show up in the sample. For example, if 13 loci were targeted, and only 9 could be reported, that would be termed, a partial profile…Over-interpretation of partial profiles can probably lead to serious mistakes. Such mistakes could include false inclusions and false exclusions, alike.” Let us examine the rape and murder of a young nurse which ultimately yielded a partial profile of the assailant many years later.

Diana Sylvester was raped and murdered in San Francisco in 1972. After evidence in this case was subjected to forensic DNA analysis, a trawl through California’s DNA database identified a suspect over thirty years later, a cold hit. Of the investigations treated here, the Sylvester murder is the one in which the particulars of the investigation (a cold hit) and the differences in expert opinion on the correct statistical calculation have the least relevance to the Knox/Sollecito case. However, several more subtle points in common can be found.

In this case the suspect was identified on the basis of his DNA matching a partial profile from evidence taken from the victim. This suspect, John Puckett was in the DNA database because of his convictions for rape about five years after Ms. Sylvester was raped and murdered. However, the MO of the crime was consistent with another rapist, Robert Baker, who matched the physical description of the assailant better than Mr. Puckett and who was deceased by the time the case was reopened. The defense was barred from introducing information about Mr. Baker, the original suspect. Over the objections of the defense, the prosecution was allowed to bring in Mr. Puckett’s past convictions. How strong was the DNA evidence against Mr. Puckett?

The main issue in this case is a disagreement among experts over the correct statistical calculation to use in cold hit cases. Another key element in this case is that it made use of a partial, not full, DNA profile. In the United States a full DNA profile has thirteen separate markers (loci). A profile must have seven markers for it to be searched in California’s database. However, only 5 and a half markers were clearly found in the Sylvester murder. The jury was told that the chances of a random person’s matching the DNA found at the crime scene were 1.1 million to one. Yet when the odds were calculated with a different set of statistical assumptions the odds were only one in three, a statistic that the jury was barred from hearing. This is why the odds were only 1.1 million to one, and not substantially higher. However, when one uses a model that takes into account that there were 338,000 profiles in the database, one arrives at the 1-in-3 odds. The question of which model is better is a difficult one, yet it is odd that California courts have taken it upon themselves to decide which model is more appropriate. And the difference between one in 1.1 million and 1 in 3 is huge. It would have probably been enough to move at least one juror from guilty to not guilty beyond a reasonable doubt.

A second issue concerns the number of loci in profile itself. The peaks that were needed to flesh out the profile from 5.5 loci to 8 loci fell below the laboratory’s intensity threshold, but forensic technician Bonnie Cheng wished to interpret them anyway. DNA profiles contain artifact peaks, some of which are known as blobs and others as stutters. Deciding which peaks are true and which are artifacts may contain an element of subjectivity. The prosecution’s outside DNA witness, Dr. Chakraborty used Mr. Puckett’s full profile to interpret the partial profile, a practice that is open to serious question.

A third issue is that the sample was mixed, containing DNA markers from both the victim and assailant. The question of which markers belong to which profiles naturally arises. DNA forensic expert Peter Gill said, “If you show 10 colleagues a mixture, you will probably end up with 10 different answers.” With reference to this case Dan Krane made an important point, “There is a public perception that DNA profiles are black and white. The reality is that easily in half of all cases—namely, those where the samples are mixed or degraded—there is the potential for subjectivity.”

A fourth issue is that the lab used up so much of the sample that the defense could not do its own testing. This is a debatable practice in a DNA case; some guidelines indicate that this should not be done unless the defense agrees in advance.

One lesson for students of the Knox/Sollecito case is that the judge’s decisions might control the outcome of a case. The judge’s decision to exclude one statistical model in the Puckett case can be compared with Judge Massei’s decision not to order an independent review of the forensics of the Knox/Sollecito case. A second message is that DNA profiling is not immune to cognitive bias. In both the Puckett case and the Knox/Sollecito case, the forensic technicians lowered the bar with respect to the intensity threshold. A third fact to bear in mind is that Meredith’s DNA profile from the kitchen knife was a partial profile. However, how many loci should be counted for this piece of evidence depends entirely upon one’s choice of intensity threshold. Fourth, the bra clasp is a mixed sample, and interpreting mixtures contains an element of subjectivity.

Some possible reforms
Australian criminal defense barrister Peter Faris wrote in response to the Benjamin Forbes case, “Faced with a figure of 10 billion, a jury will always convict. The only way that it can be defended is for the defence to demonstrate a doubt arising from contamination, forensic or statistical procedures. In the real world, defence lawyers and legal aid bodies are just not equipped to investigate these matters. Many cases rely (to a greater or lesser extent) on DNA evidence. In some of them, such as Forbes, there is simply no other evidence. Consequently, if there is contamination at the scene, in police or laboratory storage or in the laboratory itself, the defence can never prove it.

Mr. Faris proposed some reforms:
“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. This can be done by amending the various Evidence Acts around the country to make DNA evidence inadmissible unless the trial judge is first satisfied, by the prosecution, that it should be admitted. The prosecution should apply to the trial judge, in a preliminary hearing, to admit DNA evidence. They must satisfy the judge, on the balance of probabilities, of various matters. The important matters are that the burden of admissibility is upon the crown. No DNA evidence could be led unless this procedure has been completed.
Conditions could be imposed in the legislation to protect the accused. 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.”

Final Thoughts
We have examined the problems with clerical errors, contamination, statistical manipulation, and outright fraud in this and preceding posts. It must also be borne in mind that the presence of DNA on an object rarely provides information on the time or means (primary, secondary, etc.) of its deposition. Paradoxically, DNA profiling is the branch of forensics with the firmest grounding in science. Much evidence exists to the effect that it is held in particularly high esteem by juries (a possible subject for a future post).

Taking all of these issues into account, I would not convict someone in a case where the sole piece of evidence comes from DNA forensics, but I would give it considerable weight, provided that the data were obtained legitimately and presented to the court in a transparent manner that respected the discovery rights of the defense. The worthy question of whether or not states should demand DNA samples upon felony arrest is outside the scope of this post.

How does the Patricia Stallings case shed light on the murder of Meredith Kercher?

Part XVI in the Knox/Sollecito case

Patricia Stallings was convicted, then exonerated of poisoning her infant son Ryan with ethylene glycol, the major component of antifreeze. When Ryan was unable to keep food down, Mrs. Stallings took Ryan to Cardinal Glennon Children’s hospital accidentally, which happened to be associated with a toxicology unit. Ryan had metabolic acidosis (the pH of his blood was too low) and the laboratory associated with St. Louis University reported 180 milligrams per liter of ethylene glycol.

Ryan was put into foster care, but Mr. and Mrs. Stallings were allowed weekly visits. Four days after one such unsupervised visit, during which Mrs. Stallings gave Ryan a bottle, Ryan became ill again, having metabolic acidosis and died three days later. The same lab reported finding 911 milligrams per liter of ethylene glycol in Ryan’s serum and also in his baby bottle, which had been put into a dishwashing machine.

What are the odds of the tests being wrong?
There were three separate determinations of ethylene glycol, the original serum, the serum after the second feeding, and the bottle itself. In addition, one or both of the serum samples was also retested by a commercial lab. How could this be just chance? The prosecutor told the jury not to worry about motive, but just to concentrate on the fact that there was no rational explanation for these results except deliberate poisoning. He implied in so many words how unlikely it was for the labs to be wrong.

An alternative explanation begins to emerge
Patricia Stallings was arrested after a half-empty bottle of antifreeze was found in the couple’s basement. Mrs. Stallings was pregnant with her second son at the time of her arrest; her son David was immediately placed in foster care. David began to exhibit the same symptoms as Ryan, but he was taken to a children’s hospital. The hospital diagnosed David with a genetic disease known as methylmalonic acidemia (MMA)*. In this disease, one enzyme in a particular biological pathway is defective, causing a metabolic traffic jam, in which other compounds build up in the bloodstream. Although the odds of any one infant having this disease are about 1 in 48,000, David’s diagnosis meant that there was a one in four chance that Ryan had the disease as well. This fact was not allowed to be presented at her trial however, possibly because the judge and the defense attorney misunderstood each other with respect to what the defense attorney needed to do in order to bring the issue up.

Professor of biochemistry William Sly was one of the citizen-scientists who became involved in the case. Molecular geneticist Piero Rinaldo also reexamined the evidence, with some surprising results. James Shoemaker was involved in gathering evidence prior to the trial and subsequently. Both William Sly and Robert Ritter, Mrs. Stallings’ second attorney, learned of the case from the television show "Unsolved Mysteries.”

How could the analyses of ethylene glycol have been done better?
The gas chromatography (GC) technique measures how much time it takes for substances to pass through a column of stationary liquid (a waxlike substance) over which inert gas flows. This period is the retention time of a substance, and this length of time depends on its chemical properties. Two compounds may have the same retention time but usually do not.

The first analysis of ethylene glycol showed that the unknown compound in Ryan’s serum had a retention time within 30 seconds of a standard of ethylene glycol. A difference of 30 seconds is larger than is typically seen for an identical compound run twice, but let us assume generously that 30 seconds is within experimental error between one GC run and the next. The investigators should not have concluded that they had identified ethylene glycol even under this assumption, rather they should have performed additional tests.

One way to distinguish between two compounds that have identical or almost identical retention times is to run each compound through a mass spectrometer as it emerges from the end of the column. The mass spectrometer measures how heavy a compound is and in some cases how heavy the fragments of the compound are when the compound disintegrates. The combined technique gas chromatography-mass spectrometry (GC/MS) is usually very discriminating between one compound and the next, but it was just coming into widespread use about the time of the Stallings case in 1989.

A second way to distinguish two compounds is to run the gas chromatography experiment three times with three chemically distinct stationary phases. If the retention times of the two compounds, the unknown and the standard, are the same in all three trials, then the two compounds are probably identical. A third way is to spike a portion of the sample with the unknown compound with the reference. This eliminates the factors that cause run-to-run variation in retention times. The labs in question apparently did not do any of these three additional tests.

Shoemaker, Sly, and their coworkers examined a stored sample of Ryan’s blood using gas chromatography/mass spectrometry and showed that the substance believed to be ethylene glycol was actually propionic acid, one of the substances that would be expected to be elevated if Ryan had the same genetic disorder as his brother David. They also spiked a sample of Ryan’s blood with ethylene glycol and showed that it did not have the same retention time as the unknown compound, now identified as propionic acid.

The analysis of Ryan’s blood after the unsupervised visit suggested a very high concentration of ethylene glycol. Yet ethylene glycol is highly water-soluble and clears the body with a half-life of 3.0 hours. The half-life is the time it takes for the concentration of a substance to go from some value x to x/2. Therefore, Ryan would have had to consume 300 liters of ethylene glycol during his unsupervised visit to account for his blood plasma concentration four days later, an absurd volume.

Dr. Rinaldo found evidence of methylmalonic acid in Ryan’s serum. Along with propionic acid, methylmalonic acid would be expected to be in elevated concentrations in a person suffering from MMA. He also reexamined the GC data from the two labs. He found that the commercial lab saw an unknown compound in Ryan’s serum and merely assumed it was ethylene glycol without even running a standard.

He was unable to find evidence of ethylene glycol in the tests that were run on the bottle, but the bottle itself had gone missing. The positive result for ethylene glycol on the baby bottle should have raised a red flag on its own. Ethylene glycol in the bottle would have been washed and rinsed away in the dishwasher. Drs. Shoemaker and Sly believed that the small amounts of ethylene glycol found in some of the samples were due to contamination.

Cognitive bias
Once some of these facts emerged, D.A. McElroy joined with Mrs. Stallings’ new lawyer in a motion of ineffective counsel. First Mrs. Stallings was granted a new trial, and later the charges were dropped.

The first analysis to show ethylene glycol was done in a toxicology laboratory, and they might have been trained to focus on poisonings, not inherited metabolic disorders. Dr. Bob Smith called this an “expectation effect,” and he later referred to the general forensic phenomenon as “investigator bias.” The commercial lab that retested on of the serum samples may have been guilty of confirmation bias. It seems likely but not a certainty that the second lab knew that ethylene glycol poisoning was suspected. In this context it is interesting that Dr. Shoemaker later sent normal plasma spiked with propionic acid to three laboratories and two of the three reported finding ethylene glycol.

The lab that first identified the unknown compound might or might not have been following the standard protocols of the day, but the protocol was insufficiently rigorous in the sense that the identification was only one piece of information, the retention time, as discussed above. However, the commercial lab was deficient by any measure. Clearly, neither lab did a thorough job of examining all information pertaining to Ryan’s serum.

Roger Koppl discussed many of the problems that beset forensic science in the United States. Some of the problems he illuminates, such as dependency bias, were not factors in the Stallings case but may have been at work in the Knox/Sollecito case. He wrote, “the typical forensic worker has psychological incentives to reach findings based on his
close psychological ties with the law enforcement community he serves.” He also treated confirmation and other cognitive biases (p. 13).

“Larry S. Miller demonstrates an excellent example of cognitive bias at play. He asked a group of 14 students trained in hair analysis, all of whom met the basic requirements for expert testimony on human hair identification in courts of law, to examine four cases each. For each student, two cases were presented the usual way: They were given two samples and told that one was from the crime scene and the other from the suspect. The other two cases were presented through a forensic lineup. The known sample from the imaginary crime scene was compared to five suspect-known hair samples. In all 56 cases, there were in reality no true matches. The first group of cases yielded an error rate of 30.8 percent; the second group an error rate of only 3.8 percent.12

Miller’s study illustrates how evidence is often presented to the forensic scientist in a bias-inducing manner. The samples are labeled as coming from the defendant or from the victim and are frequently accompanied by a synopsis of the investigation indicating the reasons that the investigators believe the suspect is guilty. This protocol cues the forensic worker to the expected or correct result.”

The Stallings case in the context of the Knox/Sollecito case
The Johnson/Hampikian open letter criticized both the DNA evidence surrounding the knife and the bra clasp. Some observers of the Knox/Sollecito case have asked what are the odds that both of the DNA results could be the result of contamination or secondary transfer. One might ask what are the odds of at least four identifications of ethylene glycol being wrong, and yet they were in Stallings case. It may be that assigning a probability to the chances of both the bra clasp and the knife being the result of contamination or secondary transfer by standard rules is misleading. When two events are independent, the probability of both occurring are the product of the probabilities of each. So if event A has a 10% probability, and event B has a 10% probability, then the probability of both is 1%. However, when some form of cognitive bias is in play, then this calculation does not apply; in other words, the two events are no longer independent.

Some other parallels can be found. In both cases the investigators seemed oblivious to the contradictory nature of their findings. The second determination of ethylene glycol in Ryan’s serum was so high as to rule out ethylene glycol poisoning four days earlier. The lack of blood on the kitchen knife should have told the forensic DNA analysts that Meredith’s DNA profile was the result of contamination. In both cases the motive for the crime was dubious. Patricia Stallings was thought to be guilty of Munchausen’s by proxy syndrome on the basis of scant evidence. Amanda Knox was accused of trying to get back at Meredith Kercher, because of tensions from rooming together.

References
Michelle Hoffman, “Scientific Sleuths Solve a Murder Mystery,” (1991) Science 253, 931.

Roger Koppl, “CSI for Real: How to Improve Forensics Science”

Dr. Bob Mead, “Accused and Convicted—a case study”

Journal of Pediatrics, The. 1992 Mar;120(3):417-21. Misidentification of propionic acid as ethylene glycol in a patient with methylmalonic acidemia. Shoemaker JD, Lynch RE, Hoffmann JW, Sly WS.

Bill Smith, “Not Guity! How the system failed Patricia Stallings”

Appendix: Methylmalonic acidemia* (MMA)
A metabolic pathway is a series of chemical conversions, in which an enzyme catalyzes each reaction. Enzymes are the products of genes, and an alteration of a gene from its normal form may produce an enzyme that does not function properly. The compound propionyl coenzyme A is the thioester of propionic acid conjugated with coenzyme A (CoA), a thiol. When propionyl coenzyme A is produced in the body, it is converted into succinyl coenzyme A in a pathway that is three steps long: Propionyl CoA carboxylase is the enzyme that changes propionyl CoA into D-methylmalonyl CoA, and this compound is converted into L-methylmalonyl CoA by methylmalonyl CoA epimerase. Finally, this compound is converted into succinyl CoA by the vitamin B12-dependent enzyme methylmalonyl CoA mutase. The gene for this last enzyme is defective in methylmalonic academia, and the inheritance pattern is autosomal recessive. When this happens, the previous compounds build up in concentration. At some point the bond linking propionic acid or methylmalonic acid and coenzyme A is hydrolyed, and the free acids are found in the bloodstream. Among the symptoms of this disease are lethargy, seizures, and vomiting. Metabolic acidosis and ketosis are also observed. Some forms respond to supplementation with vitamin B12.

References to Appendix
Healthline
Nelson, DL and Cox, MM, "Lehninger Principles of Biochemistry," 5th edition.

*update 6/3/10, 4:30 PM. Corrected several spelling errors/typos.

Secondary transfer of DNA and DNA contamination

Part XV in the Knox/Sollecito case

Introduction
This post returns to the subject of forensic DNA contamination. It will define primary and secondary transfer of DNA and explain how they are related to contamination. It also treats clerical errors and the need for independent review of the forensic DNA data. It reconsiders the lessons of the Leskie case. These concepts will be applied to some of the DNA forensics in the murder of Meredith Kercher, especially as it relates to the evidence against Amanda Knox and Raffaele Sollecito.

DNA contamination
People sometimes confuse DNA contamination with secondary transfer of DNA. Let us define these terms and give a few examples. In Forensic DNA Typing, p. 152, John Butler wrote, “Contamination implies the accidental transfer of DNA.” Implicit in his definition is that the transfer happened after the evidence was collected. In An Introduction to Forensic DNA Analysis, p. 14, Norah Rudin and Keith Inman “define contamination as the inadvertent addition of an individual’s physiological material or DNA during or after collection of the sample as evidence…A contaminated sample is one in which the material was deposited during collection, preservation, handling, or analysis.”

Primary transfer of DNA
According to forensic scientist Suzanna Ryan, “Primary transfer is described as the transfer of DNA from an individual to an item.” Ryan summarized some of the factors that contribute to how much DNA is transferred: “Through studies by Ladd, et al and others, many in the forensic community believe that some individuals just naturally shed more cells when handling an item (‘good’ shedders) than others (‘poor’ shedders). Other factors including the substrate being handled (rougher items collect more DNA), the time since the individual last washed their hands, how nervous the person is (nervousness can lead to increased sweating), and how often they touch their mouth, eyes, hair, face or other body parts (thus gathering DNA on their hands) play a heavy role in whether a DNA profile will be obtained through simply touching an item.” One presumes that rougher items cause more cells to transfer than smoother items, but Ms. Ryan did not give specific examples.

Secondary transfer of DNA
Suzanna Ryan wrote, “In secondary transfer, there is no direct contact between a person and an object. It is instead transferred through an intermediary. This could be another person or another object. An example—expanding upon the same knife example as above—is as follows: Person A shakes Person B's hand. Person B touches the handle of a knife. Secondary transfer theory would allow that Person A's DNA could be transferred first to Person B's hand and second to the knife handle. This would mean that even though Person A never actually touched the knife handle, his DNA could be present on it.”

Secondary transfer at the scene and the lab
Suzanna Ryan continued, “At the laboratory, the analysts are careful to wear gloves when handling evidence items. Great care is generally taken to clean scissors, tweezers, and other utensils between testing items. But what about items that are not cleaned as regularly? For example, is it possible to transfer DNA from an item of evidence to a ruler, and then when the next item of evidence is examined and photographed, could the DNA transfer again from the ruler to this item? A 2006 study by Poy and van Oorschot showed an example of secondary transfer when a mixed DNA profile was found on a swab taken from an examination magnifying lamp. This profile was searched in the lab's database and a match was found with a case that had been worked on the bench-top with the magnifying lamp. It was determined that DNA was transferred from the item being examined to the analyst's gloves and then onto the top of the magnifying lamp.”

This incident should be considered both secondary transfer of DNA (the item transferred DNA to the glove, which transferred it to the lamp) and DNA contamination because it happened in the analysis lab. This incident also illustrates how easy accidental DNA transfer can be in a lab. DNA contamination occurs when an analyst transferred his or her own DNA onto an object, but this would be classed as primary transfer.

Sometimes DNA contamination occurs in ways that do not involve the item of evidence. DNA profiles have been observed because an item of labware (plastic tubes, cotton buds, etc.) was contaminated with DNA from a person who helped to manufacture the item. The chemical reagents and equipment needed to perform the polymerase chain reaction, the DNA amplification step, are occasionally contaminated. These are examples of contamination that are due neither to primary nor to secondary transfer.

Primary and secondary transfer when several people handle an object
In the journal Nature, 387, p. 767 (1997), R. van Oorschot and M. Jones wrote, "Objects handled by many individuals all produced profiles with multiple alleles of varying intensity. To determine the effect of multiple handlers, we exchanged polypropylene tubes between individuals (2 or 3, 10 min each) with different genotypes. Although the material left by the last holder was usually present on the tube, that of previous holders was also retrieved to varying extents. The strongest profile obtained was not always that of the person who last held the object, but was dependent on the individual. We regularly observed profiles of previous holders of a tube from swabs of hands involved in these exchanges, showing that in some cases material from which DNA can be retrieved is transferred from object to hand (secondary transfer).” This article was the first one of which I am aware to document that DNA profiles could be recovered from fingerprints. This may also be the first one to use the term “secondary transfer.”

Primary and secondary transfer of DNA to the bra clasp
Some have argued that the amount of Raffaele Sollecito’s DNA on the clasp can only have been deposited through primary (direct transfer), not contamination or secondary transfer. This argument is false on several grounds. It ignores that that the amount of DNA in primary transfer depends upon several factors (see above). It ignores that contamination can produce strong profiles that analysts have mistaken for genuine ones (see below). It ignores the fact that Mr. Sollecito’s DNA profile on the clasp was relatively weak in intensity, roughly 200 RFUs. Perhaps most fundamentally, this argument runs counter to a principle of DNA forensics. As part of his analysis of the Leskie case (see below), forensic scientist Dan Krane wrote, “In fact, it is quite uncommon for DNA tests themselves to say anything about the circumstances (or even the time frame) associated with the transfer.” Jason Gilder wrote in an email to me, “One of the standard axioms of DNA typing is, the presence of a DNA profile says nothing about the time frame or the circumstances under which DNA was transferred to that item. Contamination falls into is one such instance of an issue that cannot be identified by the electropherogram alone (unless you are dealing with a control sample or other known profile).”

On the other hand, an anonymous commenter on a discussion board offered this hypothetical scenario for secondary transfer of Mr. Sollecito’s DNA: “For example, let's say that DNA from Meredith, Amanda and Raffaele was on the hand towels Rudy took from the bathroom (highly probable, since they'd all used the bathroom in the days before the murder). Drying your hands on a hand towel is exactly the kind of rubbing action that would shed DNA. If Rudy had trodden on one of the towels, then trodden on the bra clasp (one of the hooks was deformed, of course) that could have transferred DNA from the towels to the hook, with no direct contact from Raffaele necessary. In turn, Raffaele's DNA was more easily identified because they could use the more sensitive tests for the Y chromosome, whereas the other DNA would have been mixed female and thus tricky to correctly identify.” This is a speculative scenario, but it is not the only kind of secondary transfer that one could imagine. Sollecito’s DNA was likely on the door to Meredith’s bedroom, for example, because he tried to break the door down.

Clerical errors in DNA forensics
Previously we have explored why the electronic files are important. However, simply having an independent expert review the case file can catch some types of errors even without the electronic files. DNA forensic consultant Norah Rudin encountered a case where two reference samples were switched, due to a clerical error. Lazaro Sotolusson was held for a year and would have been tried for rape involving juveniles, facing multiple life sentences. In a preliminary hearing, one of the female victims identified Sotolusson as her attacker, possibly a case of a mistaken eyewitness. Dr. Rudin examined handwritten notes and computer files (not the sort of files one uses to create a DNA electropherogram, such as a .fsa file) and concluded that the lab had switched the two samples. The lab retested samples from Mr. Sotolusson and Joseph Coppola and confirmed the error. Rudin stated to a reporter, “If I hadn't looked at the original data, I likely wouldn't have found the mistake. That's one of the reasons that I always look at the original data… I believe every case needs to be reviewed by an independent analyst.” (emphasis added)

Errors of a similar kind have occurred elsewhere. Professor William Thompson wrote, “I recently reviewed the corrective action file for an accredited California laboratory operated by the District Attorney’s Office of Kern County (Bakersfield). Although this is a relatively small laboratory that processes a low volume of samples (probably fewer than 1,000 per year), during an 18-month period, it documented multiple instances in which (blank) control samples were positive for DNA, an instance in which a mother’s reference sample was contaminated with DNA from her child, several instances in which samples were accidentally switched or mislabeled, an instance in which an analyst’s DNA contaminated samples, an instance in which DNA extracted from two different samples was accidentally combined into the same tube, falsely creating a mixed sample, and an instance in which a suspect tested twice did not match himself (probably due to another sample-labeling error).” (emphasis added)

Whether or not an independent analysis would have caught possible clerical errors here is an open question. The Knox defense team asked for an independent analysis of all of the forensic data, but the judge denied the request. As long as human beings are part of the testing process, clerical errors are likely to happen at some frequency. This factor and the possibility of contamination ought to be considered when one ponders the astronomical odds that are sometimes used when describing a particular DNA result.

The Leskie case and its relationship to the knife and bra clasp as evidence
Jaidyn Leskie was a toddler who was murdered in Australia over a decade ago. A DNA profile belonging to a Ms. P. was found on two pieces of Jaidyn’s clothing, a bib and some track pants. Ms. P’s profile had varying intensities at different loci, suggesting sample degradation. However, her profile was quite strong in some loci, having peak intensities of over 1000 RFUs. Another example of contamination occurred in State of Illinois v. Sean Evans (2003). In this example a positive control was contaminated with a sample containing a DNA ladder, a series of DNA fragments of increasing length that might be used as a calibration standard. Note that the intensity of the fragments making up the ladder is 500-1500 RFU, similar to a typical evidence sample. The unusual nature of the ladder DNA helps to show that contamination occurred, but this is seldom true for other kinds of contamination.

Ms P’s DNA profile was ultimately attributed to contamination partially on the basis that Ms. P lived hundreds of miles away from the crime and never left her village. Yet she could have lived next door to the Leskie family, and contamination would still be a likely explanation for finding her DNA on the toddler’s clothing. Jaidyn’s body was found submerged in water for months. These conditions are antithetical for the preservation of DNA evidence. Significantly, none of Jaidyn’s DNA was recovered from his clothing. This should have been a reason to suspect contamination on its own.

The bloodless kitchen knife in the Knox/Sollecito case is similar to Jaidyn’s clothing in that no human DNA was likely to have been on the blade at the time of collection at any section that tested negative for blood; therefore, the DNA was most likely deposited in the lab. The Leskie case was one in which the electronic data files were particularly helpful. The Knox/Sollecito case would also benefit by independent examination of these files. For example, one could use the .fsa files to examine other samples along the knife blade and handle using the same threshold value for all samples. One might find profiles belonging to other individuals, and this would strengthen the argument that DNA on the knife arose from contamination or secondary transfer.

Could contamination have occurred in the Leskie case? The evidence with Ms P’s DNA was examined within a few days the evidence from the Leskie case, and Dr. Krane noted “even if evidence samples from the two cases did not come into direct physical contact, secondary transfer (by way of common contact with a surface or implement) of biological material from the condom or articles of Ms. P’s clothing in the rape investigation to the bib and track pants could have occurred.” The lab in question had argued that contamination was unlikely on at least two grounds, one of which was the lack of DNA from Ms P showing up in other profiles. Dr. Krane agreed that “no other samples including negative controls associated with both investigations display indications of Ms. P’s allele’s being present.” However, he rebutted the lab’s point by stating, “there is no good reason to expect that contamination would uniformly affect all samples if and when it does occur. In much the same way, Salmonella-tainted meat placed on one part of a countertop at the beginning of food preparation does not mean that all food subsequently prepared for the same (or even a later) meal will be similarly tainted – just those items that also come in contact with the same portion of the countertop can be reasonably expected to have a chance of being tainted.” Although the amplification of DNA through use of the polymerase chain reaction (PCR) creates the possibility of contamination via the amplified DNA, Dr. Krane did not think it is as likely in this case as secondary transfer.

There is one more point with respect to the bra clasp that needs to be addressed. It is sometimes argued that Mr. Sollecito’s DNA could not have arrived on the clasp by contamination because his DNA was found on no other items except for a cigarette butt. This is essentially the reasoning of Judge Micheli, who oversaw Rudy Guede’s trial. From the Wikipedia article on the murder of Meredith Kercher, “The judge at the trial of Guede acknowledged that the DNA sample was considered small, but described the claim of contamination at the laboratory as making ‘no sense’, since there was no material from which such contamination could have come, and so ‘the risk would have been the loss of traces found there, not the risk of somehow discovering new traces.’” This argument is false for at least two reasons. It ignores the possibility of contamination from Mr. Sollecito’s DNA in the lab, and it ignores the empirical facts of the Leskie case, where Ms. P’s DNA did not show up in other items or controls.

Summary
One way to distinguish between secondary transfer and contamination is to say that the difference between primary and secondary transfer depends upon how the DNA was deposited on the evidence item, but contamination depends upon when the DNA was deposited: if the DNA were deposited before the item was collected as evidence, it would not be contamination. We have also seen that contaminating DNA does not evince a difference in intensity that would allow it to be distinguished from DNA arising from primary or secondary transfer before the item was collected as evidence. Clerical mix-ups are another source of error in DNA forensics, and catching them is one of many reasons to welcome independent reviews of the results. Raffaele Sollecito’s DNA could have arisen on the bra clasp from contamination or secondary transfer, as well as primary transfer. Meredith Kercher’s weak, dubious DNA profile on the knife is almost certain to have arisen from contamination.

Updates 1/13/2012 and 1/19/02

I corrected Suzanna Ryan's name from Suzanne, and I fixed a link to one of her articles. I added a quotation mark and fixed a link to William C. Thompson's article.

The prosecution's failure to release the electronic data files to the defense

Part XIV in the Knox/Sollecito case

(updated three times on 4/27/10, once on 5/6/10, and once on 5/20/10)

One of the first things that drew me to the Knox/Sollecito case was the open letter on two items of evidence, the kitchen knife and the bra clasp. The two coauthors, Drs. Elizabeth Johnson and Gregory Hampikian are forensic DNA scientists. A one-paragraph appendix to the open letter was co-signed by seven additional experts in the forensic DNA field, including Dan Krane and Jason Gilder. The open letter completely dismissed the DNA evidence linking the knife to the crime and said. “Handling and movement of this sample [the clasp] has compromised its probative value.”

Dr. Johnson was initially approached by someone close to the defense team about looking into this case, but I have no information on how the other individuals became involved. The defense gave Drs. Johnson and Hampikian the limited materials that were released in discovery; however, the other seven did not have these data, owing to time. The seven co-signers wrote, “…While I have not had the opportunity to review all of the case-specific data in this matter, I agree that the scientific evidence that they describe does not convincingly associate the kitchen knife with the murder of Meredith Kercher. I also agree that the handling and movement of the bra clasp that they describe would have compromised its probative value, and that the presence of a DNA profile does not speak in any way to the time frame or the circumstances under which DNA became associated with this sample…” The seven names appear at this website but not on the pdf file of the letter itself; however, several of them have confirmed their involvement in the case in email correspondence.

One portion of the open letter made specific mention of the lack of release of the electronic data files. Drs. Johnson and Hampikian both requested the release of the electronic files used to construct the DNA electropherograms (for one manufacturer, these are called .fsa files), according to Jason Gilder. Dr. Krane did so on multiple occasions, according to Jason Gilder, who is one of the seven co-signers of the open letter. Dr. Krane had previously put electronic data files to very good use in the Leskie case; therefore, it is no surprise that he would request them here. The requests were made via the defense lawyers, but the prosecution steadfastly refused the requests. According to Dr. Krane, the release of electronic files is the almost universal norm. Dr. Norah Rudin provides on her website a standard form, DNA discovery request, for cases involving DNA forensics. From this document:
“2. Items #7 and #8 listed in the Discovery Request refer to electronic data, also known as raw data.
a. Electronic data is considered standard discovery and is critical to an independent review.
b. Electronic data must be received prior to commencing an independent review.
c. The laboratory will typically provide you with a CD containing electronic data.”

According to Amanda’s stepfather Chris Mellas, the defense team had asked for the DNA forensic data (this would be expected to include the electronic files and machine logs) but was told that they did not exist. Then the defense learned that the Kercher family’s lawyer had some of this information and demanded that the judge order the release of the data in the summer of 2009. The prosecution released some data, but not what was asked for. The lack of release of these data was one of the reasons for a mistrial motion in the fall of 2009. In April of 2010 Bob Graham wrote, “It has also emerged that the prosecution has failed to deliver to the defence all the paperwork and documentation related to the forensic testing. Chris Mellas, Knox's step-father who is currently in Perugia, said yesterday: ‘Our lawyers asked for everything, every file and record relating to the forensic testing. We were given some of the stuff, like what was on Meredith's shoes or a juice glass but not the full reports on the knife used or the bra-clasp.’ Deputy prosecutor Manuela Comodi brushed off the request for all forensic documentation and added: ‘They have everything they need. That is enough.’” Ms. Comodi’s words imply that the defense did not receive everything, just what the prosecution claims is enough.

The news reports from last summer and fall are consistent with Mellas’s recounting of the events. The defense did not know until last July that Patrizia Stefanoni had written the words, “Too low” on files relating to the DNA profile culled from the knife. ABC news reported in September of 2009, “The trial reopened with an attempt to have the case thrown out, but it was rejected after the judge and the jurors deliberated for 90 minutes. Lawyers for Knox and Sollecito became animated in their assertion that evidence had been withheld from them. Sollecito's lawyer, Giulia Bongiorno, addressed the court for 20 minutes arguing that the defense was not provided with crucial details of Sollecito's DNA allegedly found on Kercher's bra hook until July 30, 2009. The rights of the defense were damaged, she said, when ‘documents regarding the quantity of biological material on the bra hook and documents regarding the procedure used to attain DNA results were not made available to the defense.’”

Sara Gino, a defense expert witness, still did not have the dates on which the samples were run as of September of 2009. Frank Sfarzo reported in October of 2009 that the defense also asked the judge for an independent review of the forensics, which was not granted. Bob Graham reported in December of 2009, “Other forensic experts in several countries – including two from Britain – have started to study the DNA results but have delayed giving a verdict until they have received precise details on the methodology used by their Italian counterparts. The failure of the prosecution to provide these details to Knox and Sollecito’s defence teams is one of their central complaints.”

Do the defense team and the experts it consults have a right to obtain the data in electronic form? Release is clearly routine in the United States, consistent with the principle of transparency, enunciated by Keith Inman and Norah Rudin. Good science does not change when one crosses the border between nations. As an anonymous DNA forensic expert speaking about paper copies of the DNA forensic data said, “Certain parameters have irrevocably been applied to the data; as such it represents an interpretation by the laboratory rather than the original data.” A defendant should have the right to see and to challenge the evidence against him or her, and that principle is at risk in the Knox/Sollecito case.

Update I, 4/27/10: I corrected a minor typographical error in the first paragraph, where Dr. Johnson's name appeared twice.

Update II, 4/27/10: According to Chris Mellas by the time the defense learned that Mr. Maresca, the attorney representing the Kerchers, had some of the DNA forensic data, they had already requested it twice, before the pretrial and before the trial itself. Their third request in the summer of 2009 resulted in a court order that was not followed. To the best of my knowledge the additional DNA experts associated with the open letter made their requests after this time.

Update III, 4/27/10: Joy Halverson and Marc Taylor have confirmed that they signed the appendix to the open letter. Marc Taylor wrote, “We have discovered numerous manipulations of the data analysis or the actual physical analysis of the evidence by reviewing the electronic data…The electronic data is clearly the ‘best evidence’ in the legal system.”

Update IV, 5/6/10: In response to my query to another person who signed the letter, Simon Ford confirmed that he signed the letter and replied, “In my opinion, it is always important to review the electronic data underlying a test result, if only to confirm the integrity of the data set. In low level DNA cases such as this, in which the slightest contamination could compromise the test result, it is absolutely essential to review the electronic data for the key samples and the associated controls to check for low level contamination. I cannot think of any valid reason why the prosecutor would chose not to provide the electronic data. In my experience, forensic DNA analysts in the US are invariably happy to turn over the data to support their findings.”

Update V, 5/20/10
The Irish Times - Saturday, February 27, 2010
DNA testing project offers fresh hope to the wrongfully convicted

A forensic genetics expert, Dr Hampikian’s expertise is called on by the Innocence Project all over the US and further afield. He is currently working with the family of Amanda Knox, the American student jailed in Italy last year for the murder of British student Meredith Kercher.

A professor of genetics at the University of Boise, Idaho, Dr Hampikian’s role with the Innocence Project is voluntary, but one he takes seriously. He has been involved in four exonerations over the past decade, though, ironically, his DNA testing has confirmed guilt in two further cases.
“I can’t tell who is telling the truth or who is lying, but the DNA can tell,” he says. “Sometimes the Innocence Project is a bit of a misnomer. In two of the four exonerations, I developed evidence that led to the arrest of somebody new for the crime, so for some people we are the Guilty Project – and that’s an important part of what we do.”

This documents that Dr. Hampkian, like Dr. Johnson, is a consultant for the Knox defense team.