Saturday, January 30, 2010

Meredith Kercher’s Bra Clasp

Part V in a series on the Knox/Sollecito case

Meredith Kercher’s bra clasp was separated from her bra, presumably by the murderer. The first problem with the bra clasp in this case is the fact that it was not collected as evidence until about 47 days after the crime, on 18 December 2007. This might not matter if the crime scene had been secure. However, someone had moved the clasp had from its original location (by a meter in some estimates). Someone had broken into the house on two separate occasions in early 2008, after the clasp was in police custody. The lack of control over the bra clasp alone might be enough to disqualify it as evidence.

A second problem is that the clasp became dusty because it was found in a pile of clutter. Human epithelial cells are a major component of dust; indeed, dust itself has recently become of forensic interest (http://www.newscientist.com/article/mg19826584.200-telltale-dna-sucked-out-of-household-dust.htm). A third problem with the clasp is that it was handled with dirty gloves. Here is a video that documents this fact, as well as the dust on the clasp itself:

http://www.youtube.com/watch?v=gLE4s3jXTVU&feature=related

The glove might have touched the same door or door handle that Raffaele Sollecito used when he attempted to break the door down after Meredith’s murder. This is one possible route of secondary DNA transfer. In addition, note how it was treated in a second video:

http://www.youtube.com/watch?v=UMaTI0SiuLw

The clasp should have been handled with disposable tools. Moreover, the clasp is being passed to and fro unnecessarily, and even put on the ground, both of which increase the likelihood of contamination. One wonders why the clasp is being put on such an elaborate display.

The mixture of DNA

The clasp had Meredith’s profile as the strongest component. A relatively weak component (200 relative fluorescence units) appears to be Raffaele’s DNA. There are reports of DNA from at least three other people (http://www.dailymail.co.uk/news/article-1234298/Amanda-Knox-The-troubling-doubts-Foxy-Knoxys-role-Meredith-Kerchers-murder.html

The 19 November 2009 letter coauthored by Drs. Hampikian and Johnson said, “Transfer of Raffaele’s DNA to the clasp could have occurred through several innocent means as a result of his DNA being in the apartment or via Amanda’s clothing or belongings. DNA testing cannot determine how biological material was deposited onto an item of evidence: whether by direct deposit, or by secondary transfer through an intermediary. DNA testing cannot determine how long biological material may have been on an item, or whether contamination occurred during collection.”

http://www.friendsofamanda.org/files/KnoxSollecitoDNAPetitionSubmitted11.19.09b.pdf

The presence of a mixture leads to several questions. First because I cannot envision how so many people could have handled the bra clasp directly, I think that indirect transfer of DNA is the more likely explanation. Second, it is difficult to see why the three individuals who deposited DNA on the clasp should not be considered potential suspects. Third, suppose that the police had taken other dusty items into evidence at the same time as the bra clasp. If these had been utterly clean of DNA, the results on the clasp would carry more weight. From what I can gather, no such items were taken into custody on the same day.

The lack of other DNA

At a pretrial hearing Mr. Sollecito’s defense team argued that the lack of his DNA on the bra itself is significant (http://www.telegraph.co.uk/news/3255101/DNA-on-Meredith-Kerchers-bloodied-bra-was-due-to-lab-contamination-claims-Sollecito.htmlhttp://www.telegraph.co.uk/news/3255101/DNA-on-Meredith-Kerchers-bloodied-bra-was-due-to-lab-contamination-claims-Sollecito.html), “But defence lawyers used a mannequin's bust and a white bra to argue that it would be impossible for Mr Sollecito's DNA only to have ended up on the tiny clasp and not the rest of the bra if he had touched it or handled it.”

The Hampikian/Johnson open letter said, “Neither Raffaele Sollecito’s nor Amanda Knox’s DNA was found on: the remainder of the bra that was found with the victim, other items of victim’s clothing, objects collected from the room where the victim was found, or in samples from the victim’s body. These evidentiary samples were all collected the day the body was discovered.”

The lack of Sollecito’s or Knox’s DNA on anything in Meredith’s room except the bra clasp is hard to square with an attack that the prosecution claims to have lasted at least 20 minutes.

Conclusions

The main issues I have with the clasp are that it must have been handled by at least one unknown person, that it picked up dust during the 47 days, and that there are deposits of DNA from other individuals on it.

The Johnson/Hampikian letter deserves the last word: “Handling and movement of this sample has compromised its probative value. The laboratory results for this sample cannot reliably be interpreted to show that the DNA of Raffaele Sollecito was actually on the bra clasp at the time of Meredith Kercher’s murder, and it does not establish how or when this DNA was deposited or transferred.”

Sunday, January 24, 2010

Forensic DNA contamination

Part IV in a series on the Knox/Sollecito case

Before we examine the bra clasp in the murder of Meredith Kercher, we need to explore the problems of forensic DNA contamination. This background may also help to explain the problems relating to the DNA sample culled from a kitchen knife discussed in part I of this series.

Definition

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.” When this is done deliberately, this constitutes evidence-tampering, although there is no bright line between tampering and contamination.

Proper collection of evidence

Dick Warrington has written two useful articles on contamination for Forensics magazine. He stresses the importance of changing gloves frequently and using disposable tools. If disposable tools are not available, one must clean them between handling different pieces of evidence. In the 2009 article he cautions, “If you pick up one piece of evidence and then pick up another piece of evidence you can transfer evidence from the first item to the second item. You can avoid this kind of cross-contamination if you remember to change your gloves before handling each piece of evidence.”

Sources of contamination

The polymerase chain reaction (PCR) technique is used to increase the amount of (amplify) DNA by a maximum of 2n times, where n is the number of cycles of PCR. Because n is typically greater than twenty, this process amplifies the amount of DNA in every sample by over one-millionfold.

In Forensic DNA Typing, p. 152, John M. Butler notes “Contamination implies the accidental transfer of DNA. There are three potential sources of contamination when performing PCR: sample contamination with genomic DNA from the environment, contamination from samples during preparation, and contamination of a sample with amplified DNA from a previous PCR reaction (Lygo et al. 1994). The first source of contamination is largely dependent on sample collection at the crime scene and the care taken there by the evidence collection team (see Chapter 3). Environment contamination can be monitored only in a limited sense by ‘substrate controls’ (Gill 1997). The latter two sources of contamination can be controlled and even eliminated by using appropriate laboratory procedures and designated work areas (see Chapter 4).”

Footnotes for Butler

Gill, P. (1997) Forensic Science International, 85, 105-111.

Gill, P. and Kirkham, A. (2004) Journal of Forensic Sciences, 49(3), 485-491.

Lygo, J.E. et al., (1994) International Journal of Legal Medicine, 107, 77-89.

Dr. Donald Riley writes, “It is often said that the most critical source of PCR contamination is DNA from previous PCRs. Again, a PCR produces many DNA copies of the target DNA sequences. Due to shear number, these copies (called amplicons) are a hazard for future PCRs… However, a more dangerous source of contamination is what is called genomic DNA. This is DNA that hasn't yet been amplified. Genomic DNA doesn't have the high concentration of the target DNA copies but is a hazard because genomic DNA could produce an entirely false DNA profile. Full profile contaminants have been documented on multiple occasions and in multiple laboratories. Partial profile contaminants are more common and sometimes constitute a poorly recognized risk in using partial profiles in evidentiary samples as evidence. When contamination occurs there is rarely any way to confirm how it happened.” (emphasis added) The wonder of the PCR technique, its ability to make almost unlimited quantities of DNA, is also its danger.

Preventing and detecting contamination in the lab: the limits of negative controls

Donald Riley delineates some parallels between sterile technique and the techniques used in DNA forensics lab to minimize contamination. However, he also notes that clinically sterile equipment or reagents might still harbor DNA and that DNA samples lack an immune system to ward off contaminants. Moreover, good technique does not ensure a zero probability of contamination. Hence, there is a need for negative control experiments.

Terri Sundquist and Joseph Bessetti of Promega Corporation write:

A ‘reagent blank’ control consists of all reagents used during sample processing but contains no sample. This control is used to detect DNA contamination of the analytical reagents used to prepare the sample for analysis. In a separate negative control reaction, water is used instead of extracted sample or reagent blank. This negative control reaction is often referred to as the ‘no-template’ control and allows identification of contamination in the amplification reagents themselves.”

However, Donald Riley noted some deficiencies in negative controls, “Alternatively, the blank may show no profile, consistent with, but not proving that contamination didn't occur. Unfortunately, a few forensic DNA laboratories omit their controls…Good PCR technique is no guarantee that contamination didn't influence the results. Steps must be taken to try and detect contamination. Negative controls are blank PCRs that have all the components of the evidentiary PCRs but have no other DNA added intentionally. Fortunately, there are often two negative controls used, one when the DNA is extracted, and another when the PCR is set up. Any PCR signal in the negative control would warn that contamination has occurred. Unfortunately, the negative controls are virtually the only warning of PCR contamination. Negative controls may alert the analyst to general contamination occurring within the lab or the lab reagents. These controls don't offer protection against contamination occurring before the samples arrived at the PCR lab. Negative controls also can't rule out contamination of individual samples. The individual samples lack individual signs of contamination if it occurs. Unlike a human patient, a PCR is incapable of showing signs of infection (contamination) such as fever or undue pain. PCRs also have no immune system to ward off contaminants.”

Butler, pp. 152-154, implicitly recognizes the problems associated with the PCR amplification process:

“The possibility of laboratory contamination is assessed with ‘negative controls’ that test for contamination of PCR reagents and tubes. Basically, a negative control involves running a blank sample through the entire process in parallel with the forensic case evidence. The same volume of purified water as DNA template in the other samples is added to a negative control PCR reaction. If any detectable PCR products are observed in the negative control, then sources of contamination should be sought out and eliminated before proceeding further…To reduce contamination problems during the laboratory examination of DNA samples, all pre-PCR and post-PCR amplication reactions should be kept physically separate. Laboratory contamination is probably impossible to avoid completely, but can be proactively assessed with negative controls and staff elimination databases (Gill and Kirkham, 2004).”

Norah Rudin and Keith Inman (p. 15) state: “Once the sample is in the laboratory where it can be dried and chilled, the potential for contamination is mostly from other samples undergoing processing at the same time…Precautions include processing evidence and reference samples separately in space and time, restricting PCR product to an isolated room, and using controls to detect contamination in any batch of samples.” (emphasis added)

But William C. Thompson is clear about the limitations of negative controls,

“A false incrimination can also occur through cross-contamination among evidentiary samples. Even labs that are careful to test reference samples separately from evidentiary samples often process all of the evidentiary samples from a case together, creating the potential for false matches. I recently reviewed a case processed by the Los Angeles Police Department DNA laboratory in which samples from a bloody murder scene were being processed in the same batch as samples from items collected in a suspect’s house. Due to an analyst’s error in the case, DNA from the murder victim accidentally ended up in a control sample. This error was detected because the control sample was a “blank” which was supposed to contain no DNA. However, it was merely happenstance that the accidental transfer of DNA ended up in a blank control rather than another sample in the same batch. If the victim’s DNA had instead ended up in one of the samples from the suspect’s house, I believe that the error would not have been detected and would have led to a false laboratory report saying that the murder victim DNA had been found on an item collected in the suspect’s house. Defense lawyers need to think carefully about the potential for such errors because experience shows that they can and do occur.” (emphasis added)

Riley concurs, writing, “These controls don't offer protection against contamination occurring before the samples arrived at the PCR lab. Negative controls also can't rule out contamination of individual samples. The individual samples lack individual signs of contamination if it occurs.” The bottom line is that some instances of contamination probably go undetected.

Dishonesty involving controls

William C. Thompson delineates another problem:

“While most of the problems are due to inadvertent mistakes, a number of cases involving dishonesty have also come to light. 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.”

Footnotes for Thompson

14. See, U.S. Department of Justice, Office of the Inspector General, The FBI DNA Laboratory: A Review of Protocol and Practice Vulnerabilities, May 2004. Available online at: http://www.usdoj.gov/oig/special/0405/index.htm

15. Laura Cadiz, Md.-based DNA lab fires analyst over falsified tests, Baltimore Sun, Nov. 18, 2004.

16. Author’s interview with Robert Shaler, former Director of the OCME DNA Laboratory.

17. Associated Press, Worker in Army lab may have falsified DNA test result. Aug. 27, 2005; Memorandum For All Staff Judge Advocates Re: Brady Notice by Lisa Kreeger, Staff Attorney, Department of the Army, October 17, 2005 (on file with the author).

When the investigators and the DNA laboratory are in communication, the investigators sometimes make it clear that a certain result is desired. Thompson and colleagues (2003) write:

“Part of the problem is that forensic scientists refuse to take appropriate steps to ‘blind’ themselves to the government’s expected (or desired) outcome when interpreting test results. We often see indications, in the laboratory notes themselves, that the analysts are familiar with facts of their cases, including information that has nothing to do with genetic testing, and that they are acutely aware of which results will help or hurt the prosecution team. A DNA analyst in one case wrote:

‘Suspect-known crip gang member — keeps ‘skating’ on charges-never serves time. This robbery he gets hit in head with bar stool — left blood trail. [Detective] Miller wants to connect this guy to scene w/DNA …’

In another case, where the defense lawyer had suggested that another individual besides the defendant had been involved in the crime, and might have left DNA, the DNA laboratory notes include the notation: ‘Death penalty case. Need to eliminate [other individual] as a possible suspect.’”

The problem of having the laboratory be independent from the prosecution is a well-known one (http://learn.genetics.utah.edu/content/labs/gel/forensics/): “The [National Research Council] recommends that forensic DNA analysis be conducted by an unbiased outside laboratory that maintains a high level of quality control and a low error rate.”

Case Studies of Contamination from Co-Processing of Samples

Thompson gives two examples of suspect identification on the basis of dubious cold hits:

“In one particularly interesting Australian case, DNA on the clothing of a murdered toddler named Jaidyn Leskie was linked, via a ‘cold hit,’ to a young ‘mentally challenged’ woman who lived hundreds of miles away and who, by all accounts, had never left her own village. Police could find no way to link the young woman to the Leskie murder and at first dismissed the ‘cold hit’ as an ‘adventitious’ (coincidental) match. However, a coroner’s investigation established that DNA from the young woman had been processed through the same laboratory at about the same time as the toddler’s clothing. The young woman had allegedly been the victim of a sexual assault involving a condom. Although laboratory personnel maintain that accidental transfer of samples between cases is impossible, it now appears almost certain that the young woman’s DNA from the outside of the condom accidentally contaminated samples from the toddler’s clothing.

“The facts of some recent cases in the United States have also raised suspicions about false cold hits due to contamination across cases. For example, in 2002, while investigating the 1969 murder of University of Michigan law student Jane Mixer, the Michigan State Police Crime Laboratory in Lansing found DNA of two men on her clothing. The profiles were searched through a database and matched two Michigan men, Gary Leiterman and John Ruelas. Police immediately suspected that Leiterman and Ruelas had been involved in the murder, but there was a problem — Ruelas was only four years old when Mixer was killed and had been living with his parents in another city. According to news accounts, police could find no link between young Ruelas and Mixer.29 That did not deter Washtenaw County Assistant Prosecutor Steven Hiller who charged Leiterman with the murder. Hiller ‘created a scenario placing a young Ruelas at the [murder] scene as a chronic nose-bleeder whose blood dropped on Mixer.’30 There is, however, another possible explanation for this ‘cold hit.’ Examination of laboratory records revealed that known samples of DNA from both Leiterman and Ruelas were being processed in the Michigan State lab on the same day as the old samples from the Mixer murder.31 Both men were being tested in connection with other cases unrelated to the Mixer murder. Although the Michigan State laboratory maintains that cross-contamination of samples across cases was impossible, it seems a very strange and unlikely coincidence that two men who, according to the prosecutor, were present when Mixer was murdered in 1969 just happened to have their DNA tested (for other cases) on the very same day as samples from the Mixer case were tested. Leiterman was nevertheless convicted of Mixer’s murder in 2005.” (emphasis added)

Footnotes for Thompson

29. Maryanne George, Murder case mystery deepens. Detroit Free Press, Jan 15, 2005.

30. According to news accounts, Hiller offered no evidence to support this theory. Liz Cobbs, Judge raises possibility evidence may have been contaminated at State Police lab, Ann Arbor News, May 11, 2005.

31. Author’s interview with Professor Dan Krane (a defense expert in the case). Also, Thersa Mask, Mixer’s dad is clear on one thing, Detroit Free Press, July 13, 2005.

Contamination is surely an unlikely event, at least in that it happens less frequently than it does not happen. Yet, which is less likely in these two cases, contamination or the involvement of the suspect identified by these cold hits? And even if improbable, how does this speak to a reasonable doubt?

Borderline situations

Suppose that a technician runs evidentiary samples and reference samples in the same batch out of laziness or ignorance. Suppose a different technician runs these samples together knowing that there is a greater chance of contamination. These two cases are distinguishable only if one knows the intent of the technicians. The second case is close to outright evidence tampering, but the similarity of the two situations implies that there is no bright line between sloppiness and outright fraud.

Frequency of Contamination

This is difficult to estimate for a variety of reasons. Donald Riley reports that some laboratories omit controls or perform them in such a way as to lessen the chances of detection. Thompson reports that many laboratories fail to record instances of contamination in a log book, despite the advice of the FBI. Some labs take the overly sanguine approach that if they detect contamination, it is not really an error. Thompson writes, “The surprise for defense lawyers who have managed to gain access to these files is how voluminous they are. Errors occur regularly.”

Summary

  • Forensic DNA contamination is a well-known phenomenon that good labs attempt to minimize, and to document when it does happen.
  • PCR-amplified DNA is one of the most serious contamination threats in a DNA forensics lab.
  • Keeping a clean DNA lab is a little like maintaining a sterile environment, only more difficult.
  • It is difficult to estimate the frequency of contamination for a variety of reasons. However, many instances have been documented.
  • Negative controls can be used to identify contamination, but they only detect contamination in blanks, not evidentiary samples.
  • Fradulent DNA forensics often takes the form of falsifying negative controls.

Bibliography

John M. Butler, Forensic DNA Typing (2005), 2nd ed., Elsevier.

Terri Sundquist and Joseph Bissetti

http://www.promega.com/profiles/802/ProfilesinDNA_802_11.pdf

Donald Riley

http://www.scientific.org/tutorials/articles/riley/riley.html

Nora Rudin and Keith Inman, An Introduction to Forensic DNA Analysis, 2nd ed. (2002).

William C. Thompson 2006

http://www.nacdl.org/public.nsf/0/6285f6867724e1e685257124006f9177

William C. Thompson; Simon Ford; Travis Doom; Michael Raymer; Dan E. Krane (2003)

http://www.nacdl.org/public.nsf/698c98dd101a846085256eb400500c01/c1d63d2bd1a582cf85256e540074c146?OpenDocument&Highlight=0,rape

Dick Warrington Forensics April/May 2005

http://www.forensicmag.com/articles.asp?pid=41

Dick Warrington Forensics April/May 2009

http://www.forensicmag.com/articles.asp?pid=275

Tuesday, January 19, 2010

Occam's razor versus Knox's behavior

First, kudos to Observer for some fine links. I especially liked http://lesswrong.com/lw/1j7/the_amanda_knox_test_how_an_hour_on_the_internet/ and I hope that everyone takes the time to read the postscript. Second, the comments thread for the previous post on Dr. Kekule has drifted into a discussion of Knox’s behavior. That’s OK; I was considering hosting an open thread on her behavior/credibility anyway. So, let’s keep using the Kekule thread for questions of behavior and try to stay more on topic in future forensics threads. Comments are disabled for this very brief post.

Update (22 January 2010)
I will consider submissions on the subject of the Knox/Sollecito case. Here is your chance to be a guest blogger.

Saturday, January 16, 2010

Commentary on the Knox and Sollecito case from Dr. Alexander Kekule

Professor Alexander S. Kekule wrote following commentary for the German newspaper Der Tagesspiegel on 9 December 2009 (http://www.tagesspiegel.de/meinung/kommentare/Amanda-Knox-DNA-Spur;art141,2970520). Professor Kekule holds both M.D. and Ph. D. degree and is a virologist. I am very grateful to two anonymous friends, who did most of this translation.


Amanda Knox - on ice angel’s trail
The verdict against the “icy-eyed angel” has met with criticism, especially in the US. Actually, the evidential value of DNA tests is over estimated.

Last Friday one of the most controversial murder trials of recent years ended. A jury in Perugia, Italy sentenced 22-year-old Amanda Knox, and 25-year-old Raffaele Sollecito to 25 years imprisonment. The Court regarded it as proven that the American student and her boyfriend in the town of Puglia, Italy in November 2007 brutally abused and murdered a fellow student. Another accomplice, 23-year-old Rudy Guede, in a separate case, was already sentenced to 30 years. The conviction of the "icy-eyed angel" met with criticism, especially in the U.S., because the evidence was allegedly insufficient.

Under Italian law, the judge must justify the verdict within three months following the trial. However, if the reports of the process observers are correct, already the prosecution relied mainly on forensic evidence. The version presented by the prosecutor, who in the court appears to have followed essentially something that sounds bizarre, but not impossible: during the night of the crime, Knox was to have pushed her British flatmate Meredith Kercher to organize a sort of belated Halloween orgy with the two men. When she refused, Sollecito held her tightly, Guede raped her, and Knox cut her throat.

The evidence against the Ivorian Guede, who has a criminal record, appears overwhelming. Traces of his sperm were found on the dead woman, his finger and footprints in her blood. Afterwards he fled to Germany. However, he denies the murder and accuses Knox. The evidence is more scant against the American woman and her boyfriend. Knox herself came under suspicion because she claimed to have first seen a Congolese bar owner at the scene. Meanwhile, she claims not to having been at home at all that night, and to have found the corpse only the next morning. Evidence of her and her boyfriend’s finger- and footprints in the shared home are no proof since both were frequently in the apartment. Also, the smashing of the window from the inside, presumably executed by the perpetrator to fake a forced entry, could have been the doing of Guede, whom Knox and Sollecito accuse of the crime.

All this would hardly suffice for a conviction - if there were not three tiny DNA traces. Sollecito's genetic material was found on a bra closure of the victim, which had apparently come loose during a violent opening. And in Sollecito's apartment, investigators found a kitchen knife with a traces of the victim's DNA on the blade and Knox’s DNA on the handle.

Indeed, confusion of individuals through "genetic fingerprinting" is almost impossible. It examines not the actual genes in the DNA (they are quite similar in all humans), but the useless segments between them (satellite DNA). Because it carries no genetic information, satellite DNA is copied sloppily and often repeatedly during the formation of egg and sperm. This results in regions of repetitive DNA, the repeat pattern of which is unique for each person. The likelihood of confusion is smaller than for fingerprinting.

But the forensic DNA analysis has its pitfalls. While a real fingerprint shows that the person has actually touched an object, DNA evidence may arise in many different ways. In the Italian murder case, the DNA on the bra clasp could also have originated from a skin scale, lost by Sollecito days earlier in the apartment. The DNA traces on the knife blade could have been transmitted through the hands of Amanda Knox, who lived together with the victim and used her boyfriend’s knife for cooking. The evidence would have more weight if in addition blood stains were found.

All three convicted have appealed the convictions. In that case, the DNA traces have to be re-evaluated, because the presumption of innocence also applies to ice angels. As the mother of the victim has rightly said: "At the end of the day only evidence counts, nothing else."

The author is Professor of Medical Microbiology and Virology at Halle, Germany, and Director of the Institute for Medical Microbiology. Photo: J. Peyer

Sunday, January 10, 2010

Ominous Parallels: Comparing the Duke lacrosse case to the Knox/Sollecito case


Amanda Knox and Raffaele Sollecito were convicted of murdering Meredith Kercher, Amanda’s flatmate in Perugia, Italy, in 2009. Both Ms. Kercher and Ms. Knox were studying abroad. Ms. Kercher was from Great Britain, Ms. Knox is from Seattle, Washington; Mr. Sollecito is an Italian student. A third suspect, Rudy Guede, was convicted in 2008. To some students of the Duke lacrosse (DL) case, the Amanda Knox/Raffaelo Sollecito (KS) case is playing like a sequel they did not wish to see.
DNA evidence
In the Duke lacrosse case the only DNA that even might have been from one of the three indicted players was on a fake fingernail found in a trashcan in one of the bathrooms. The DNA was a weak match to David Evans; however, the most likely explanation was that it arose from transfer from items of his in the trashcan, such as dental floss or facial tissue. In the Perugia murder case there are two pieces of evidence, the kitchen knife discussed in part I of this series, and the bra clasp, which will be treated in a future post. The kitchen knife is alleged to have DNA from Ms. Knox on the handle and a trace of DNA from Ms. Kercher on the blade (http://viewfromwilmington.blogspot.com/2010/01/amanda-knox-and-raffaele-sollecito-and.html). The bra clasp is said to have DNA from Ms. Kercher, Mr. Sollecito and possibly three other individuals. Its value as evidence is disputed.
The Prosecutor
DA Michael Nifong not only withheld exculpatory DNA information, but also made inflammatory statements to the press. His conduct was so egregious that a new verb, to Nifong, has entered the English language. Giuliano Mignini has the distinction of having had a book written about his conduct during a murder investigation, The Monster of Florence, by Douglas Preston and Mario Spezi (http://opinionator.blogs.nytimes.com/2009/06/10/an-innocent-abroad/?ref=opinion). Mr. Mignini was indicted in 2006 based upon his actions in the Monster of Florence murder case (http://www.cbsnews.com/blogs/2009/12/07/crimesider/entry5928444.shtml). Mr. Mignini denied any wrongdoing (http://news.bbc.co.uk/2/hi/europe/7883286.stm).
Changing Storyline
After Reade Seligmann’s attorneys released evidence of the younger Mr. Seligmann’s whereabouts at the time of the alleged rape, Mr. Nifong implied that he had a different timeline, a transparent attempt to get around an unimpeachable alibi. In December of 2006, the alleged victim said that she was no longer sure that she had been penetrated by a penis. Many students of the DL case saw this as an attempt to explain away the lack of players’ DNA even in the presence of DNA from other individuals in the AV’s body.
The KS case has been through more changes in storyline than a movie script handled by a dozen writers. Pulitzer Prize winning reporter Timothy Egan wrote in the summer of 2009, “‘Case closed,’ the Italian authorities said in those first days of November, 2007, even though they had yet to arrest the only man [Rudy Guede] who has ever been found guilty of the murder.” At one point the possibility that the murder was related to a Halloween ritual was floated; at the close of the trial this was not mentioned. In both the DL and RS cases the only constant was that the defendants must be guilty.
Presumption of Innocence
A prominent southern writer, Alan Gurganous, wrote an article for the New York Times titled, “Blue Devils Made Them Do It.” Mr. Gurganous said (http://www.nytimes.com/2006/04/09/opinion/09gurganus.html?ex=1302235200&en=97bf0167bd611343&ei=5088&partner=rssnyt&emc=rss), “From north of here, this story must seem like yet another involving Southern frat boys run wild.” Mr. Gurganous does no more than to pay lip service to the principle that a defendant is innocent unless proven guilty. If anyone doubts that many leading figures in Durham and elsewhere trod the presumption of innocence into the ground in this case (in other words, if one doubts that Mr. Gurganous’ view was representative), Taylor and Johnson’s book Until Proven Innocent will disabuse him or her of this notion.
With respect to the KS case “Judge Paolo Michelli, during the pretrial, took the conspiracy for granted. He boasted that he began his reasoning with all three suspects in the murder room. So much for innocent until proven guilty (http://blog.seattlepi.com/dempsey/archives/179047.asp).”
Character assault
Nifong gave about seventy interviews in the first weeks of the DL case. Among his many false assertions was that the players were stonewalling the investigation. The Durham police aided and abetted him.
One of the criticisms leveled against Ms. Knox is that she failed to act appropriately after the murder. Attorney Scott Greenfield posted an essay on the judicial weakness of such an argument (http://blog.simplejustice.us/2009/06/11/how-is-she-supposed-to-act.aspx). Greenfield also wrote (http://blog.simplejustice.us/2009/12/05/a-trial-without-evidence.aspx), “During the summation, the prosecutor told the jury about the things Amanda Knox might have said to Meredith Kercher before the alleged drug-induced orgy that ended with her throat being slashed. ‘You are always behaving like a little saint. Now we will show you. Now we will make you have sex.’ This would be a horrible thing to say, except that it never happened. No one says that such a statement was ever made. But summations in Perugia aren't limited to evidence, as they are here. Rather, this is a permissible indulgence into fantasy, a made up dramatization of what the prosecutors contend might have happened.” Indeed, many of the incidents that have been used to paint Ms. Knox in an unfavorable light are either untrue or misleadingly portrayed, and may be the subject of separate posts.
Irrelevant or false leaked information
Unindicted sophomore Ryan McFayden made an ill-judged allusion to the book and movie “American Psycho” in a private email. Virtually everything written about the hard-to-stomach contents of this email had to do with the character and mental health of its author (I have firsthand knowledge, however, that Ryan McFayden is a polite, thoughtful, and intelligent young man). Too few people have pointed out that the email had nothing to do with the case and that the authorities were wrong to release it.
Amanda Knox’s journal, which was written during her pretrial incarceration, was leaked to the press. Apparently, it had been translated into Italian then back-translated into English. Along the way the meaning changed in ways that were unfavorable to her (http://www.sciencespheres.com/2009/11/crucible-of-perugia.html). Moreover, upon being informed that she was HIV-positive (which was not true), she wrote about her sexual partners in her journal. This information has contributed to her public image as a person of low sexual morals. However, the ethics of leaking her journal have received scant attention. Some of the leaks in the K/S case, such as the claim of receipts for bleach (http://www.timesonline.co.uk/tol/news/world/europe/article2894139.ece), were not presented at the trial and are probably false (http://www.friendsofamanda.org/cleanup.html).
Wendy Murphy
Among the pundits who got the Duke lacrosse case wrong, Wendy Murphy ranks close to the top (http://durhamwonderland.blogspot.com/2006/12/wendy-murphy-file.html). She made numerous factual errors, and speculated wildly and with an unchecked anti-player bias. Her defense of DA Michael Nifong was bizarre (http://liestoppers.blogspot.com/2006/12/wendy-murphy-strikes-again.html).
Students of the DL case might expect similar behaviors with respect to the Knox/Sollecito (KS), and Ms. Murphy does not disappoint (http://www.patriotledger.com/opinions/x1682953943/WENDY-J-MURPHY-Is-Foxy-Knoxy-an-innocent-coed-or-manipulative-murderer). She wrote,” DNA on the handle of the knife that killed the victim matched Knox - and blood on blade matched the victim…Pro-Amanda forces also forget to note that the knife was found hidden in a shoebox, far back inside a closet at Sollecito's apartment - and that the knife had been scrubbed clean with bleach and an abrasive substance - like a Brillo Pad. The defense claimed the sample of Knox's DNA was too small to matter, but ANY DNA is damning evidence - especially on a knife that's been intentionally cleaned and hidden away deceptively in a shoe box, tucked deep inside the closet of a suspect's home.”
Let us focus only on her factual errors about the knife, the subject of Part I of this series (http://viewfromwilmington.blogspot.com/2010/01/amanda-knox-and-raffaele-sollecito-and.html). There was no blood on the blade, and this is one of the main reasons to doubt that DNA signals arising from when the knife was swabbed did not really originate with the blade at all (http://www.sciencespheres.com/2009/10/lcn-dna-profiling-part-ii-watch-where.html). The knife was found in a kitchen drawer with other knives. The police stored it in a shoebox. Ms. Murphy does not explain how she divined that Ms. Knox and Mr. Sollecito used bleach to clean the knife, nor does she bother to tell us that bleach is so effective at destroying DNA that it is routinely used in DNA labs for that very reason. If bleach and a brillo pad were used to clean the knife, it is very difficult to see how any DNA could remain. Finally, the amount of DNA observed was so small that to say it matched Ms. Kercher’s profile is a stretch; calling it a partial match is a better description, and it is quite possible that it originated from Ms. Kercher’s DNA in the laboratory itself.
Courtroom Demeanor
In Until Proven Innocent Taylor and Johnson wrote (p. 180) about Collin Finnerty facing a wall of photographers while awaiting his first appearance before a judge: “He tried to remain expressionless. In that atmosphere, if he had smiled the media would have called it a smirk; if he had frowned they would have called it an angry glare.” Nevertheless one sportswriter wasn’t sure if the younger Mr. Finnerty’s expression was one of shock and fear or or smugness (http://www.usatoday.com/sports/columnist/saraceno/2006-04-18-duke_x.htm).
Frank Sfarzo wrote (http://perugia-shock.blogspot.com/2009/11/migninis-rage-against-knox-and.html) wrote of Amanda Knox, “If she smiles it's wrong, if she cries it's wrong, if she moves it's wrong, if she's still it's wrong, if she watches it's wrong, if she doesn't watch it's wrong."
The role of the blogosphere
In the Duke lacrosse case the blogosphere was able to go into issues in greater depth than the mainstream media and correct a number of important errors. Here is a partial honor roll: Bill Anderson, CrystalMess, Durham in Wonderland, John In Carolina, LaShawn Barber, Liestoppers, and TalkLeft. In the Amanda Knox case, Dr. Mark Waterbury (http://www.sciencespheres.com/2009/10/seven-deadly-sins-of-knoxsollecito.html) and Candace Dempsey (http://blog.seattlepi.com/dempsey/archives/129925.asp) have been covering forensics and general aspects of the case, respectively. Ms. Dempsey may have been the first to point out the similarities between the DL and RS cases.
Final Thoughts
This post is intended as an overview of some of the problems in the Knox/Sollecito case. Any one of these points could easily be expanded into its own post. We will try to explore some of them in the coming months, especially those involving forensics.
Part II in a series.

Update (22 January 2010)
Seattle, WA (USA), near where Amanda Knox's family lives, and Perugia, Italy are sister cities. Maybe Durham, NC should be Perugia's sister city, instead.

Sunday, January 3, 2010

Amanda Knox and Raffaele Sollecito and the murder of Meredith Kercher, Part I

Introduction to the case

This post will be the first in a series of articles on the Amanda Knox/Raffaele Sollecito case. Amanda Knox is an American student, and Italian Raffaele Sollecito is her former boyfriend. Both of these two individuals were convicted of the murder of British student Meredith Kercher in late 2009. Rudy Guede was previously convicted of her murder in a separate, fast-track trial in 2008. Many in the United States have criticized the prosecutor’s summation (http://blog.simplejustice.us/2009/12/05/a-trial-without-evidence.aspx) and the forensics (http://www.sciencespheres.com/2009/10/methods-of-polizia-pseudoscientificaa.html).

The DNA forensics in this case centers around two items, Meredith Kercher’s bra clasp and Raffale Sollecito’s kitchen knife. This article will focus on some of the issues surrounding the knife. The kitchen knife had Amanda’s DNA on the handle, which is not surprising given that Amanda cooked at Raffaele’s house. But the prosecution claims that their forensics investigators found a small amount of Meredith’s DNA on the blade. This claim is dubious at best, but even if it were true, the knife cannot be the sole murder weapon. Let’s take up the second question first.

Problems with the knife

First, Mr. Sollecito’s kitchen knife was too large to have made two of the three wounds on Ms. Kercher’s body. The smaller knife, the one that made the first two wounds, may have also made the third wound. Second, the kitchen knife does not match the bloody outline of a knife at the crime scene (http://www.newsweek.com/id/216903/page/2). Third, Newsweek reported that, “an officer testifying at the trial said he used ‘police intuition’ when choosing that knife from Sollecito's cutlery.” If there were multiple knives in the drawer, why choose one that had a different outline from the one whose outline was on Ms. Kercher’s sheets? The claim of police intuition does not make any sense.

But possibly the most serious reason for doubting that DNA was really on the knife is that it tested negative for blood (http://www.sciencespheres.com/2009/12/why-knife-was-not-knife.html. Dr. Stefanoni opined that the knife had been cleaned with bleach. This is a puzzling claim even at the outset, because bleach does not leave a corrosive mark on stainless steel; however, the prosecution’s argument becomes even more questionable upon further inspection. Drs. Elizabeth A. Johnson and Greg Hampikian, both experts in DNA forensics, coauthored an open letter about some of the evidence in this case (http://www.friendsofamanda.org/articles.html). About the cleaning of the knife, they said:

This DNA does not originate from blood. A highly sensitive chemical test for blood was negative, and 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.

Indeed, bleach is so effective at destroying DNA that it is used in research laboratories for that very purpose (A. M. Prince, L. Andrus PCR: How to kill unwanted DNA, Biotechniques, Vol. 12, No. 3, 358-360). It far more effective than acid! Bleach is also used in some anthropological work to destroy unwanted DNA on the surface of an object that would contaminate valuable DNA inside. The very fact that there was no blood on the knife suggests that there was also no DNA on the knife; therefore, contaminating DNA is the most likely source for the signals that were observed. These three reasons call into question whether the kitchen knife had anything to do with the murder.

What the laboratory of Dr. Patrizia Stefanoni should have done is to save some of the material for a retest. As mentioned previously, this lab should have tested other knives, and perhaps other, random implements, in Raffele Sollecito’s drawer in the same way. Would DNA have also shown up on a different knife? These control experiments would have clarified whether Meredith Kercher’s DNA was really on the knife or not. However, we can at least examine the data that were produced from the knife.

Introduction to DNA forensics

Modern DNA forensic analysis (birg.cs.wright.edu/talks/Human Identification 2008.ppt) produces fluorescent signals that must be observed and interpreted properly to exclude or not exclude a person as a DNA contributor. The fluorescent signals arise from a collection of DNA molecules of various lengths (sizes) that are produced by chopping the DNA strands at a set of specific locations, and then separated using capillary electrophoresis. The pattern of signals from the DNA form what is called an electropherogram. The electropherogram from a piece of evidence is compared against reference samples from various individuals.

Fluorescence Spectroscopy

Spectroscopy is the study of how light interacts with matter. All spectroscopic experiments involve the observation of photons of light above a background of random noise. All forms of spectroscopy must contend with the fact that signal-to-noise (S/N) ratios are not infinite. The S/N ratio is a measure of the strength of the signal and is often related to the concentration of the chemical species that produces it. DNA forensics makes use of chemical labels that produce fluorescence. In a DNA electropherogram the S/N ratio is measured in relative fluorescence units (RFUs). Why are S/N ratios so important in regards to the DNA sample on the knife blade in the Amanda Knox/Raffaele Sollicito case?

The open letter coauthored by Dr. Elizabeth and Professor Gregory Hampikian and cosigned by several others had this to say about the knife DNA:

An extremely low level, partial DNA profile was developed for the blade swabbing using the Identifiler kit. The alleles detected were consistent with the DNA of the victim. The highest peak in the electropherogram was approximately 100 relative fluorescence units (rfu), while 21 of the 29 peaks that were detected and labeled as alleles fell between 20 and 50 rfu….No credible scientific evidence has been presented to associate this kitchen knife with the murder of Meredith Kercher. (emphasis added)

Signal-to-Noise Ratios

To understand their conclusion we need to know more about signals and noise. Suppose you are driving your car away from your home town and you have tuned in your favorite FM radio station. As you travel farther away, the music gets fainter (the signal is now weaker), so you turn up the gain (amplification). Now the music is more audible, but you start to hear crackles (the noise). Amplification affects both signal and noise. Eventually, you will travel so far away that the noise becomes more unpleasant and you switch to a different station (the S/N ratio has become unacceptably low).

Now let us ask what would happen if you were 200 miles away and you tuned to the frequency of your favorite FM station in your home city. You would hear nothing but noise, sometimes called static. Based only on your observation, you could not say that the station was even broadcasting. It is not that you would deny that the station was broadcasting, it is just that you cannot affirm it on the basis of your observation; your observation is indeterminate.

Each DNA forensic laboratory establishes a threshold value for the size of acceptable peaks, but the threshold values are not identical from lab-to-lab. Setting a minimum threshold does not automatically favor the defense or the prosecution. The lowest such value of which I am aware is 40 (http://www.bioforensics.com/articles/champion1/champion1.html). At first this seems large, since peaks smaller than this are still greater than noise, but these peaks are small relative to those typically encountered in DNA electropherograms.

Instead of examining the specific choice of a threshold value, let us discuss why setting them in advance of the experiment is so important. Scientists set up their experiments to test (falsify) their hypotheses. If the signal-to-noise level in any spectroscopic experiment falls below the threshold, any hypothesis requiring that the signal be above the threshold must be rejected. The reason one sets the threshold first is to avoid bias. The textbook An Introduction to Forensic DNA analysis, 2nd ed. (Rudin, N. and Inman, K., CRC Press 2002, p. 121) states (emphasis added), “It is important to have some predetermined limit to distinguish what is signal and what is noise.” If one treats the threshold as flexible, the very purpose for setting it prior to running the experiment is defeated.

How does this relate to the DNA electropherogram of the knife sample (http://www.friendsofamanda.org/articles.html, Figure 1)? 16 out of 29 peaks are lower than 40 in S/N, and 22 are lower than 50 RFU. Dr. Stefanoni herself wrote (http://www.thedailybeast.com/blogs-and-stories/2009-09-14/no-mistrial-for-knox/) the words “too low” with respect to the knife. In other words these peaks are too small to count as music in the car radio analogy above.

It would be helpful to know the threshold value regularly employed in Dr. Stefanoni’s lab, but it could have been as high as 150 RFU and is very unlikely to be below 40 RFU. The signals above the threshold might constitute a partial profile of Ms. Kercher’s DNA. However, such a partial match is very problematic (http://www.scientific.org/tutorials/articles/riley/riley.html):

1. A partial profile essentially proves that one is operating outside of well-characterized and recommended limits.
2. Contaminating DNA usually presents as a partial profile, although not always. For this reason, the risk that the result is a contaminant is greater than for samples that present as full profiles.
3. A partial profile is at risk of being incomplete and misleading. The partial nature of it proves that DNA molecules have been missed. There is no way of firmly determining what the complete profile would have been, except by seeking other samples that may present a full profile.

Conclusions

The kitchen knife cannot be the sole murder weapon, and it most likely had nothing to do with the crime whatsoever. The only evidence that it might have played any role is the finding of DNA that is at best a partial match to Ms. Kercher’s DNA on the blade, and that almost certainly came from contamination during the test. Given the weakness of the fluorescence signals and especially the lack of blood on the knife, the signals observed do not tie the knife to the crime. No wonder that the open letter quoted above ends its section on the kitchen knife by concluding that there is no forensic evidence to link it to the crime. But an equally troubling problem with the prosecution’s theory is that Ms. Knox and Mr. Sollecito would bother to take this knife from his flat to the dwelling occupied by Ms. Knox, Ms. Kercher, and their two roommates in preference to using knives found in the kitchen of these four roommates. What sense does that make? It is typical of this case that Occam’s razor is so often ignored.

Update, 01/18/2010

The prosecuation's theory would also have us believe that Knox would not dispose of the supposed murder weapon, and despite cleaning the blade extensively, would not clean her fingerprints from the handle. This strikes me as unlikely.

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