DNA does fly, and it also transfers quite easily

Part 37 in the Knox/Sollecito case

Update (11/7/2013)
Poy and Van Oorshot wrote, "To further help evaluate the above finding swabs were taken from gloves worn whilst examining a heavily soiled dress during routine casework examination. A significant amount of DNA was retrieved which exhibited a genetic profile that matched that of samples taken from the exhibit."  This is direct evidence that gloves can transfer DNA.

Introduction

The issues of possible DNA contamination and proper versus improper handing of evidence are at the center of the forensic evidence in the trials of Amanda Knox and Raffaele Sollecito.  This entry will examine two aspects of DNA forensics, dealing with airborne DNA and the need to change gloves when handling a fresh item of evidence.  Both kinds of potential problems need to be recognized and addressed in order to minimize the chances of contamination.

The problem of airborne DNA

Barbie Nadeau wrote, “…the defense claims that the crime scene was badly compromised during the collection of evidence.  Alberto Intini, head of Italy’s national forensic team, disagrees. On the stand, he defended the forensics work and stressed that the crime scene had not been contaminated, especially under cross examination when the defense lawyers tried and failed to prove otherwise. ‘DNA does not fly around like pollen,’ he said…” Ms. Nadeau left her readers with a misimpression.  Later in the trial the defense returned to this issue.  “’DNA does not have wings, but it flies,’ [defense expert witness Sarah] Gino cautioned. ‘In a laboratory where hundreds of samples are examined, the risk of contamination exists and should be taken into consideration.’”

Dr. Intini’s view is seriously in error, as can be ascertained by a number of lines of evidence.  A paper on DNA in fossils (BioTechniques 38:569-575, April 2005) notes, “These molecules are easily spread via aerosol transport. One aerosol droplet can contain many more DNA molecules than one gram of fossil material.” In a discussion of how to collect DNA evidence Dick Warrington advised, “Next, you can prevent contamination by wearing a mask, since you want to avoid coughing and sneezing around the evidence you are processing.”

pipettors and pipet tips

Pipettes are devices used to deliver small volumes of liquids.  They are used frequently when the polymerase chain reaction (PCR) technique is used to amplify tiny amounts of DNA.  For that reason the use of aerosol barrier pipette tips is routine in labs engaging in PCR.  An application note on pipetting explained, “Cross-contamination occurs if improper pipetting causes splashes or drips. Even if pipettors are handled properly, aerosols can be generated that contain DNA molecules and can contaminate the pipettor and subsequent pipet products. This demands increasing efforts to reduce the penetration of contaminants by means of filter tips.”  Gilson, a manufacturer of pipettes, supplies a technical bulletin that reads in part: “For example, if a technician in a crime lab performs PCR on a blood sample, cross-contamination between samples could result in an erroneous incrimination, even if the technician changes pipette tips between samples. A few blood cells could volatilize in the pipette shaft, stick to the plastic of the pipette, and then get ejected into the next test sample. Modern laboratories have taken account of this fact and are devoting tremendous efforts to avoid this problem through the use of filter tips."

laboratory traffic

A number of forensic guidelines are in place to protect against aerosol DNA from one source or another.  For example,  “A ‘one-way traffic’ rule is also observed in the laboratory, once the technician has entered the PCR or the post-PCR rooms, they are not allowed to return to the extraction or pre-PCR rooms until the next day or a complete cloth changing in order to prevent contamination by aerosol particles.”  The problems only get more serious in the low template region of analysis.  Keith Bedford said, “The way I am speaking at the moment, we could probably detect DNA on this pad in front of me.”

laboratory design

Laboratories are constructed in a way to minimize the possibility of contamination due to air flow.  In the article Setting Up a PCR Laboratory.” Theodore E. Mifflin discussed how the design of the laboratory can minimize the chances of contamination: “Air handling. For extremely high-performance PCR laboratories that will be involved with detecting very-low-prevalence DNA or RNA molecules (e.g., infectious disease agents in clinical samples), additional measures may be necessary to prevent contamination from the air being recirculated between the pre- and post-PCR laboratories. In this case, the air handlers need to be separate and the air pressure individually adjusted in each laboratory. In the pre-PCR laboratory, there should be a slight positive pressure compared to the air in the connecting hallway. The post-PCR laboratory, in contrast, should be at slightly reduced pressure to pull air in from the outside and thereby prevent escape of amplicons from the completed PCR samples being analyzed inside the lab (Fig. 2). Finally, the air handlers for the pre- and post-PCR laboratories need to be connected to separate air ducts, and each must lead to a separate location for exhaust.”  Dr. Mifflin’s main focus is pathology, but his points about low levels of DNA are germane to low template DNA forensics in that both situations use PCR to amplify very small quantities of DNA. 

What the police did and did not do with respect to changing gloves

When the forensic police collected evidence at Ms. Kercher’s flat, they did not change their gloves frequently.  One can observe in a series of photographs a failure to change gloves over several minutes and several evidence samples.  Barbie Nadeau reported, “Sollecito’s attorney, Giulia Bongiorno, stopped the crime scene video several times to point out errors. For example, Stefanoni testified that she had changed gloves according to official investigation procedures, but Bongiorno stopped the crime scene video twice to show that Stefanoni’s bracelet and the fold of her glove were exactly the same before and after the time she claimed to have changed gloves.”  Ms. Nadeau recounts the same incident on p. 133 of Angel Face, her book on the murder of Meredith Kercher.

Ms. Stefanoni’s views on when gloves should be changed are found in the English translation of the Massei report.  On p. 203 Massei wrote that Stefanoni "specified" that gloves were changed "every time an object was touched that was particularly soaked with blood, and when it was obvious that the gloves would be soiled;"  Based on pages 204-205, she appears to believe  that the presence of a liquid is necessary to bring about contamination by touch.

What forensic experts say about changing gloves

On page 38 of John Butler's 2005 textbook Forensic DNA Typing, he wrote, "Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence."  Dick Warrington is the author of some articles in Forensic Magazine and is employed by a company which makes equipment for crime scene investigations.  He wrote, “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.”  He also advised, “Put on gloves, use gloves, change gloves. Do that every time you touch a piece of evidence. Likewise, use disposable tweezers, scalpels, etc. Change these each time they are used, as well.”  Orchid Cellmark’s guidelines for collecting DNA evidence read in part, “Use clean latex gloves for collecting each item of evidence. It is recommended the gloves be changed between the collection of each item of evidence.”

Conclusions

The need to use fresh gloves when handling a new piece of evidence is utterly noncontroversial.  Yet it is obvious from video of the collection, that the forensic police did not change gloves frequently.  Their failure to follow the consensus view of what is good practice might have contaminated the bra clasp and might have led to mixed DNA samples elsewhere in the flat.  The fact that airborne DNA can compromise an experiment is likewise the consensus view of practitioners of PCR.  Dr. Intini’s ignorance of the dangers of aerosol DNA shake one’s confidence in the ability of a forensic lab under his supervision to combat the dangers of airborne DNA contamination even in routine DNA profiling, let alone low template profiling.

Unresolved issues in the latest round of DNA testing in the Hank Skinner case

DNA testing in the Hank Skinner case began again in 2012 after the state of Texas dropped its objections after a fight that lasted over a decade.  After the release of some preliminary data, the prosecution declared that it confirmed Skinner’s guilt, as reported by Jordan Smith in the Austin Chronicle.  Mr. Skinner’s attorney issued a response stating that not all testing was complete and that defense experts had not had a chance to examine the results yet.  Skinner had been found guilty of murdering his live-in girlfriend Twila Busby and her two grown sons.  He claims that he is innocent, and there is an alternate suspect in this case, Robert Donnell, who was Twila’s uncle.

Mr. Skinner’s DNA was found on 10 of 40 items tested.  Many of the items tested appear to have been blood, and they come from various locations around the house.  There are profiles that belong to an unknown individual or individuals, including one unknown profile on a knife that has DNA deposited by Mr. Skinner and by Elwin Caler, one of Ms. Busby’s sons.  There is also an unknown profile from a bloodstain in the sons’ bedroom.  The unknown profiles may not be complete, but that is one of several issues that further testing might resolve.

My conclusions at this time are that the advisory paints an incomplete (therefore misleading) picture of the case and is overinterpreting both the previous DNA results and the present ones.  For example, the prosecution’s advisory summarized the testimony of a witness who claimed that Mr. Skinner threatened her, but the witness later recanted, a fact that was ignored.  The advisory failed to mention anything about two bloody handprints and bloody bootprints that have been discussed in one report on this case. "The State tested the bloody handprints against those of Hank Skinner.  Hank was good for the three handprints near the back of the house.  The handprint on the trash bag, and presumably the handprint on the front storm door, belonged to someone else." The existence of the jacket, the handprints that apparently do not match, and the bloody bootprints that are not his size are some of the objections to the prosecution's case.

With respect to DNA profiling the first problem is that the most compelling piece of exculpatory evidence, what one person identified as Donnell’s jacket, has been lost before it could be tested.  The jacket is stained with blood and sweat.  If either Donnell’s DNA were found on the jacket or if some of the blood were Twila’s), that would establish reasonable doubt, at the very least.  However, at present there is no reference profile of Robert Donnell (see also below).  The failure to obtain his profile could be a consequence of the investigatory tunnel vision that has plagued this case, as a previous entry here indicated.  I suggest that the State of Texas exhume his body or obtain his DNA in some other way.

A second problem centers around whether Twila was sexually assaulted on the night of her death.  The vaginal DNA profiling was performed by standard autosomal DNA methods, in which DNA deposited by a possible perpetrator can become overwhelmed by the victim’s own DNA.  There is nothing in the public record of Y-STR testing being performed, despite its routine use in sexual assaults.  The unique advantage of Y-STR profiling is that it involves only the Y chromosome, which women do not have; therefore, a male contribution to a mixture can be selectively amplified by the polymerase chain reaction.  Either the laboratory did not do Y-STR testing, or it did but the results were not included in the advisory.  In addition the fingernail clippings either showed Ms. Busby’s DNA, or they did not show any DNA.  Moreover, fingernail scrapings can also be tested with Y-STR forensics, which has marginally higher sensitivity than autosomal testing.  Mr. Skinner’s attorney indicated that the defense has requested additional testing; therefore, it is possible that Y-STR results will be forthcoming.

A third problem concerns the existence of DNA from at least one unknown person.  The evidence with respect to the knife appears inculpatory at first glance, yet it is undisputed that Mr. Skinner was bleeding that night.  It is likely that his contribution to the knife DNA profile arose from his blood, and one obvious possibility is that he cut himself while committing the murders. The defense should raise two alternate hypotheses.  There is chance that his profile arose from casual use of this knife around the house prior to the murder.  In addition, Mr. Skinner was incoherent according to one witness due to a combination of alcohol and codeine; therefore, his DNA could have been deposited by his possibly handling the knife at some point after the murder when he was allegedly stumbling around the house.  It is also undisputed that he was in close proximity to the victims that night; therefore, positing that he handled the knife, although speculative, is not unreasonable. 

The Skeptical Juror wrote, "Fresh blood drops were found on the sidewalk, near the front door of the house where the murders took place.  Those drops were tested.  The DNA from those drops belonged not to Hank Skinner, but to an unidentified male." The most recent DNA profiling also did not test the gauze, even though it may have been used to wipe the knife, which is odd.  I wonder whether this profile was similar to or different from the ones turned up in the most recent round of testing, and I also wonder who the DNA donor(s) is(are).  The former question might be addressed by treating the DNA from the blood drops on the sidewalk as something like a reference profile.  DNA mixtures are sometimes deconvoluted by a process known as sequential unmasking, in which a forensic worker is given information about the reference profiles in a gradual manner, only after completing a relevant portion of the analysis.  Yet even the mere existence of blood from another person calls for an explanation.  Was the blood fresh?  If so, who was bleeding besides Mr. Skinner, Ms. Busby, Mr. Caler, and Mr. Randy Busby, Twila’s other son (all of whose reference profiles are available)?

Finally, a fourth problem is that one of the samples may have been contaminated with some DNA from a laboratory worker.  This kind of event actually happens not infrequently in DNA profiling, but it does mean that the defense needs to scrutinize the negative controls and machine logs to look for other evidence of contamination.  Negative controls will detect large scale contamination that affects multiple items of evidence, but will often not detect sporadic contamination affecting a single sample, as Professor William Thompson has pointed out:  “However, the same processes that cause detectable errors in some cases can cause undetectable errors in others. If DNA from a suspect is accidentally transferred into a ‘blank’ control sample, it is obvious that something is wrong; if the suspect’s DNA is accidentally transferred into an evidentiary sample, the error is not obvious because there is another explanation — i.e., that the suspect is the source of the evidentiary DNA.”

One hopes and expects that future rounds of DNA testing will address some of these shortcomings.  Both the victims’ families and Hank Skinner deserve nothing less than a full accounting of what happened that terrible night.  However, the prosecutor brings charges in the name of the people; therefore, every citizen has some responsibility for what happens in a courtroom, including the times when there is a miscarriage of justice.

The utility of electronic data files in DNA forensics cases

Part XII on the Knox/Sollecito case

“Full disclosure allows a thorough independent review of results and thereby helps assure that the underlying scientific methods are strong and appropriate, and that the laboratory’s interpretations are fair and accurate. Until the electronic data are made available, I cannot say that I have performed a complete review of the underlying evidence in this matter.”
-William Thompson, Victoria State Coroner’s Inquest into Death of Jaidyn Leskie

In a previous post (http://viewfromwilmington.blogspot.com/2010/03/interim-evaluation-of-evidence-against.html) I wrote:

Why are the .fsa files important? Technical bulletin 40-035 from Chromosomal Laboratories, Inc., is a checklist of what the laboratory is expected to provide, and it states, “Copies of all data files used and created in the course of performing tests and analyzing data in this case, including .fsa files, if applicable. These files should include all data necessary to independently reanalyze the raw data.” In response to a question on this subject, Professor Dan Krane wrote, “The biggest concern that I personally have regarding this case is the refusal of the prosecution to provide the defense with a copy of the electronic data that underlies the DNA test results -- that is virtually unheard of world-wide today and it would be especially important to review that data in a case such as this which seems to involve such low level samples.” According to a knowledgeable source, “Each tested sample has its own file. The file contains the full electropherogram trace information along with other information about the testing conditions (e.g., date, time, injection time, voltage, temperature, current, the RFU threshold used by the analyst). If you have the electronic data, you can use the DNA analysis software (GeneScan & Genotyper or GeneMapper ID) to independently analyze the electronic data. That allows you to examine the results as closely as possible (zoom in on the electropherogram to evaluate low-level results) and establish the RFU threshold of your choosing.” (emphasis added)


The knowledgable source I quoted above is Jason Gilder, and he and Dan Krane are cosigners of the open letter from a total of nine forensics scientists that is critical of the evidence in this case. I would like to reexamine this issue with respect to how these files are useful. I will have more to say about circumstances of their being withheld from the defense in the future.

Contents of fsa files
The fsa files are created by the DNA analysis instruments of Applied Biosystems, and the program GeneScan can read them. These files contain the times of and dates on which samples were (runhttp://www.nfstc.org/pdi/Subject06/pdi_s06_m01_02.htm). They also contain the peak detection threshold in relative fluorescence units. Perhaps most importantly, they contain the electronic information needed to recreate a DNA electropherogram with the parameters chosen by the analyst.

Electronic data files and the Leskie case
I would like to examine this case more extensively, both for what it tells us about contamination and what it tells us about electronic data files. Jaidyn Leskie was a young boy in Australia who was murdered n 1997. DNA from an alleged rape victim, Ms. P, was found on two items of his clothing, a bib and track pants. There is general agreement that this was the result of contamination (http://viewfromwilmington.blogspot.com/2010/01/forensic-dna-contamination.html), despite the fact that contamination did not show up in the negative control (reagent blank or no-template) samples. It is helpful to know that Jaidyn’s body was submerged in water for several months before its discover, and these conditions are not at all conducive to the stability of DNA. None of Jaidyn’s own DNA was found, only Ms. P’s.

Professor Thompson provided a report for the Leskie case (quoted above). Professor Thompson wrote (http://www.bioforensics.com/articles/index.html), “When I review the casework of forensic DNA laboratories in the United States, I typically ask for and receive copies of the electronic data collected by laboratory instruments during DNA testing. These data show the actual results of the DNA tests. By examining these data, an independent expert can check whether the forensic laboratory interpreted its results correctly and can detect a host of potential problems that would not be apparent from examining the laboratory notes. Electronic data would be particularly helpful in evaluating the present cases. Beyond allowing a cross check of the analysts’ interpretations, it would allow a detailed independent assessment of whether there is evidence of low- level contamination in any samples.”

Professor Krane provided two reports; the supplemental report was written after he was given access to the electronic data files. With respect to the negative controls not showing contamination his first report state, “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.” In previous posts, I have noted that the amount of DNA after it has been amplified by the polymerase chanin reaction (PCR) makes it a serious potential contaminant. However, professor Krane explained why the PCR process itself was not likely to be the source of the contamination in this instance. Professors Thompson and Krane both indicate that substrate controls would have been appropriate in this case.

It is Dr. Krane’s second report that is especially pertinent to the utility of access to the electronic data. He wrote, “My subsequent review of the underlying electronic data associated with the testing performed by the VFSC during their investigation of the death of Jaidyn Leskie in particular leaves me even more convinced that the STR-DNA profile match observed between the condom of the rape investigation and the bib in the Jaidyn Leskie abduction investigation is the result of contamination.” He went on to say, “Now that I have had an opportunity to review the electronic data from the Leskie investigation I, like Professor Thompson, do indeed find low levels of signal that are consistent with Ms. P’s DNA profile and lend substantially more credence to the already compelling proposition that she is the source of the DNA associated with the Leskie bib and track pants.” Professor Krane subsequently wrote in response to one of my inquiries that the observation of these peaks “helped strengthen the case that contamination had occurred by several orders of magnitude.” (emphasis added)

Professor Krane noted that there was a systematic difference in the peak heights depending on the software one used and the analysis options one chose: “However, I thought it might also be helpful to consider the output as it was seen by the testing laboratory primarily for two reasons: 1) no-smoothing with the Macintosh versions of the software systematically makes peak heights larger; and 2) the peak heights generated in this way should be more directly comparable with the threshold established by the testing laboratory in the course of their validation studies. As Professor Thompson points out, “Most laboratories establish a peak height threshold for reporting alleles and make it a practice to ignore peaks falling below that threshold.” I, like him, feel that this practice is appropriate but that low-level signals may also contain useful information in investigations such as this one.” It is possible that peaks could fall above the threshold in the hands of one analyst and below it in the hands of another. The sizes of the peaks in the two figures in this report vary from less than one hundred RFUs to many hundreds of RFUs, documenting the fact that profiles arising from contamination can easily be mistaken for samples arising from genuine contact.

Justification for the release of electronic files in another case
An anonymous DNA forensic expert unaffiliated with the present case had this to say in a case from several years ago:
“The laboratory has provided hard copy printouts of the data that do not allow the possibility of reanalysis by an independent expert. Certain parameters have irrevocably been applied to the data; as such it represents an interpretation by the laboratory rather than the original data. Of additional concern is the fact that the documents provided by the laboratory are represented as fully complying with the discovery request.” The expert gave several areas where independent review requires the electronic data files, including.
1. Determination of the threshold. “Not all qualified experts would necessarily apply the same threshold given identical data…The possibility of false inclusions as well as false exclusions exists if real data that happen to reside below an artificial threshold are ignored.”
2. Scale of viewing. “This ability is an important element in determining whether a particular peak represents and authentic DNA fragment.”
3. Proprietary concerns. “The data itself, as the work product of a government laboratory in a criminal matter, is clearly in the public purview.”
“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.”

Electronic data files and the Knox/Sollecito case
In response to a question, Dan Krane wrote me to say, “I would like to see that data primarily for two reasons: 1) to determine the actual heights and data collection points of all the peaks (for purposes of mixture resolution and the possibility that some are associated with pull up, respectively), and 2) to calculate a limit of detection from the data in the controls so as to determine if the lab was reliably distinguishing between signal and noise in the evidence samples.”

Professor William Thompson wrote (Tarnish on the 'gold standard:' Understanding recent problems in forensic DNA testing. The Champion. 30(1):10-16, January/February 2006), “A key aspect of discovery in DNA cases is the electronic data produced by the computer-controlled genetic analyzers that are currently used to “type” DNA samples. Analysis of the computer files can not only reveal undisclosed problems and support alternative interpretations of the findings, but also, as discussed above, these files can be crucial for detecting instances of scientific fraud, such as that committed by Jacqueline Blake and Sarah Blair.” Jacqueline Blake was an employee of the FBI whose two-year record of contamination-free work was the result of dumping a portion of the negative control down the sink. Sarah Blair repeatedly substituted a file of a contamination-free blank for the actual blank.

I can think of several things I would look for using the electronic data files. The first is what peak threshold was used for the 400+ runs and whether or not it was the same threshold as the knife blade profile. The second is to zoom in on the small peaks in the bra clasp, as Jason Gilder implied. It is conceivable that the identities of the three additional people who contributed DNA to the clasp could be ascertained. The third is to examine the negative control runs for evidence of fraud, as Professor Thompson noted could be done. The negative controls are the most common place for fraud to be detected.

State of Michigan
One exception to the general rule of complete disclosure of the electronic data files was the state of Michigan (http://www.bioforensics.com/articles/Thompson_Champion_Tarnish.pdf). In 2005 the Michigan State police Issued a statement saying that, “it is the position of the Michigan State Police Forensic Science Division that any release of this (sic) data for processing with non-validated parameters is tantamount to evidence tampering.” Thompson refutes this position: “By analogy, if the police disclosed digital photos of the crime scene, it would hardly be ‘data tampering’ for a defense expert to manipulate the digital images in an effort to enhance them or bring specific details into focus…The use of proper analysis parameters might well become an issue if and when the defense decided to offer into evidence the results of its analysis of the electronic data. But to deny access to digital data on grounds that the defense might analyze it improperly eviscerates the right to discovery.” (emphasis added) I will update this post if I am able to learn whether or not the State of Michigan has changed its policy.

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.