Monday, August 19, 2013

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.

21 comments:

michellesings said...

love the article. I don't know of a SINGLE time Stefanoni even changed gloves! Much less any of the other minimum standards that should have been followed! Trust me, had she done so it would be on their little "video". The one that was a little too obvious.
And yet the poseurs cling to answers that David Balding was nice enough to answer you on-- specific questions you asked him. And you, being honest, posted those answers for the sake of organic discussion because you have nothing to hide. What I love about the innocentis is that they want truth at all cost. We want only the truth and we consider it all. Big picture type people.... and there IS a much bigger picture at stake when one considers everything, not just desired pieces of the puzzle.Those findings prove valuable.
The poseurs are so intent on proving guilt that they fail miserably at realizing how much of a fail they are when it comes to finding the whole real authentic truth. The Kercher's, I believe are in fact tormented even more than their already unbearable grieving over the painful death of their innocent young daughter....I feel this way because of what surrounds them-this filth (perverts, actually) who want to befriend them so desperately.
May this family come to see the truth soon regardless of what these psychos are doing in the name of their beautiful innocent daughter who is gone forever now. The thought of them being associated with her sickens me completely. They are clouding this poor family's minds. And yet there are many of their own British friends who believe Amanda and Raffaele are actually innocent, yet they don't have the balls to lovingly kindly tell them the truth. With friends like that who needs enemies. RIP MK now back to forensics... ;)

noel dalberth said...

Excellent Chris :) Evidence collected over a month later Dirty gloves, passing the clasp around w/o tweezers. Stef's statement: 'There is no need for me to give you any more information. I am an expert.' Trying to hide somethingStef? Barbie proclaiming the 5 spots were mixed blood, when in fact it was mixed DNA. Even Massei didnt assume that mixed DNA was equivalent to mixed blood. A sloppy investigation led to contamination.

Rick.Bonin said...

I am definitely not a forensics expert, but for heaven's sake, the risks and procedures you have highlighted in this article just seem like common sense to me. You should not even have to write this, as the Italian Scientific Police should damn well know it. When we whine about corruption, this is the type of nonsense we are talking about. I don't expect Nadeau to have a clue, but what excuse is there for the Italian authorities?

Nice article Chris, written well for the rest of us.

Dave K said...

Pass the bra clasp around? It looked more to me like they were kicking it around like a soccer ball. I think they stepped on it a couple of times, too.

And they accuse US of disrespecting Meredith's memory?

Karen Pruett said...

Thanks for this Chris, your overview of cross-contamination dangers in the collection and processing of evidence literally hits the nail on the head with regard to the the Knox-Sollecito case.

Yes, the Perugians collected a huge amount of DNA, but only some of it can be used reliably.

The Bra Clasp and Knife are definitely no where near reliable. It's a travesty that "evidence" was admitted into court because even armchair forensic experts can see it's tainted.

Colin Connaughton said...

Excellent piece, Chris. To anyone with an even partial scientific background, this is all obvious and straight-forward. To any person who does not comprehend the ease of airborn movement of molecules of any substance, I would say 'Consider any substance that you can smell'. That smell is the detection of molecules of that substance which was borne by air to arrive in your nose to be detected by your nostrils. Even hard substances such as plastic, steel, or concrete can be smelled. I see no reason for DNA molecules to be an exception to the general rule.

billy ryan said...

You have summarised all that is wrong with this case,but there is a gap between presenting it here on View-from-Wilmington,and presenting it in court in a way that the court will not be able to dismiss it and retain its credibility,Stefanoni lied and cheated to ruthlessly destroy two innocent lives,people like you Chris who wish to use forensic science for the betterment of the human race have a vested interest in exposing this phoney Stefanoni in Florence

Chris Halkides said...

From the Conti-Vecchiotti report: "Dr. Stefanoni notes that the Real Time PCR reaction was prepared on the counter without using a fume hood to guarantee the absence of contamination." On the contrary, if Ms. Stefanoni prepared her reaction in her fume hood, it would increase the odds of contamination.

From an anonymous letter about the forensics labs: "The gloves? There is no need to waster them and the recommendations are to use them to the end and to not break them.”

Anonymous said...

Sorry Chris, I am not understanding your most recent comment. Do you mean someone wrote to you and told you the lab staff have been told not to change gloves?

Well, in any case, disturbing stuff. Does this head of the lab not know any better, or is he just trying to justify what was already done? Either way disturbing.

I am wondering about the loss of data on Sollecito's laptop which I guess either he or Knox said could have alibied at least one of them.

Chris Halkides said...

Anonymous at 11:57,

There was an unsigned letter detailing some of the problems in the lab. I don't have a link handy at the moment.

Anonymous said...

The thought that ISC asserts the defendants have to "prove" the path of contamination is quite disturbing. It is supposed to work a little differently, although Italy would have its own rules about this. The prosecutors are supposed to show (1) the sufficiency of the collection procedures not to introduce contamination and (2) a trustworthy chain of custody. The tape of the evidence gathering was quite enough to demonstrate likely paths of contamination. Plus, there is a clear problem with the chain of custody. How can the PM demonstrate a secure chain of custody was maintained? As shown in the tapes, the chain was clearly broken between the initial investigation and the follow up six weeks later when the clasp was collected. Does anyone believe the crime scene was secure during the time lapse or that the clasp was found in its original condition?

"The most important aspect of evidence collection and preservation is protecting the crime
scene from the time the first officer or responder arrives until the last piece of evidence
has been noted and collected without being contaminated."
http://www.policeforum.org/library/dna-forensics/DNA%20Forensic%20Evidence.pdf

"Preservation of Evidence From crime scene to forensic laboratory to courtroom, all evidence must be inventoried and secured to preserve its integrity. Crime scene evidence admissibility in court is predicated upon an unbroken chain of custody. It is important to demonstrate that the evidence introduced at trial is the same evidence collected at the crime scene, and that access was controlled and documented."
http://www.all-about-forensic-science.com/crime_scene_evidence.html

Also, see the following academic piece from a South African on the chain of custody: http://www.academia.edu/933101/The_chain_of_custody_and_formal_admissions

And below are links to a few interesting CA cases along with a discussion of how forensic evidence was admitted in the Goldman v. Simpson case.

http://caselaw.findlaw.com/ca-court-of-appeal/1153900.html

People v Williams (1989) 48 Cal.3d 1112, 1134 (unexplained anomalies in prosecution
handling of latent fingerprint card went to weight of evidence after prosecution had made
prima facie showing that card had not been tampered with).

http://mcadams.posc.mu.edu/custody.txt
_______

Finally, below is a link to some fabulous TED talks.

http://blog.ted.com/2012/09/10/6-talks-on-the-phenomena-behind-wrongful-convictions/

Scott Fraser, Peter Donnelly and Rob Warden are particularly good. Rob Warden notes that in reviewing 70 documented wrongful convictions in Cook County, Illinois, about 1/2involved a false confession or a false accusation (notably, he analyzes both false confessions and false accusations together).

Observer

Anonymous said...

Probably, the last sentence should read, "Rob Warden notes that in reviewing 70 documented wrongful convictions in Cook County, Illinois, about 1/2 involved a false confession where the confessor was implicated and convicted or a co-defendant was implicated and convicted."

Observer

Gallagher said...

Chris,

I would consider giving up on contamination if I were you, seriously, you're just digging a bigger hole for yourself.

Ask yourself, what are the chances that two given crime samples, out of all the objects tested, just happen to get contaminated with the victim's and suspect's DNA.

Why can't you say what you want to say? That is the cops deliberately placed the DNA to frame Sollecito. What's so hard with that? I don't know whether that's right or wrong, but it certainly makes a lot more sense than all this contamination business.

Chris Halkides said...

Gallagher,

Let me summarize the most recent entry for you. Frequent changes of gloves are an essential part of good evidence collection, and the failure to change them might have led to the contamination of the clasp. Among many other possible routes, airborne DNA is a common phenomenon, and it might have led to contamination of the extraction from the knife. To use a Speedvac to concentrate liquids, one has to open the centrifuge tubes to the air, and this would have made the samples vulnerable to aerosol DNA. But equally disconcerting as these technical failures is the lack of knowledge demonstrated by Stefanoni and Intini. Why should anyone trust a lab staffed by people whose views are contradicted by multiple kinds of evidence?

Your comment fails to address any of these issues. What is more, you have made it clear that you know little about DNA forensics and have no desire to learn (despite some valuable comments from others that would help). For these reasons, it is difficult to understand why you want to take a position on the likelihood of contamination. As far as I am concerned, we will not know how many samples were contaminated until there is full release of all of the data. Moreover, there is little point in wondering whether or not the bra clasp was contaminated; by the van Oorshot definition, it was contaminated.

I have always maintained that the knife egram is probably due to contamination, and I have implied that if it had been tested in a lab designed for low template analysis, there is a good chance that no profile would have been generated. With respect to the clasp, I have also made it clear that both contamination during collection and tampering are more likely explanations than primary transfer during the crime. Therefore, even your premise is open to question.

Gallagher said...

Chris,

My comment did not address the issues raised in your post because there is really nothing much to address, and I don't mean that in a negative sense. Airborne and aerosol DNA could constitute a hazard - sure. Changing gloves after touching a sample is good, not changing is bad. We all know this and I am not disputing any of it. My point concerns the likelihood of these factors leading to Sollecito's DNA. You have already posed this question to Balding, and he answered - someone would have to deposit Sollecito's DNA on the clasp, walking in and out of the room would be unlikely to do this. Let's pretend that Stefanoni never changed gloves, let's pretend she broke every protocol in the book - it would still be extremely unlikely to cause Sollecito's DNA. Yes, I'm aware of your theory about the towels and door handles, secondary and tertiary transfer, but the facts remain it would still be a highly unlikely cause. In other words, possible, but certainly not probable.

You seem intent on doing a Mignini type hatchet job on Stefanoni - she's bad, she's dirty, from southern Italy and incompetent. You seem to think this somehow leads not only to contamination, but contamination with the very suspect, someone who hardly frequented the bungalow. Where is this all going? The Supreme Court ain't impressed. They want an explanation, and if they solicit the views of experts, I can't help but feel they would agree with Balding, that is not changing gloves at the appropriate time, dropping the clasp on the floor, not using tweezers etc. do not constitute a realistic contamination threat. You must surely be aware of this, and I can't help but wonder, why people with strong ties and links to the pro innocent lobby wish to proceed down this road. Why not just claim Sollecito's and Knox's DNA on the clasp and knife were a direct result of police fraud. They kind of dither on this question. Why not come straight out and say it? Maybe lawyers have advised otherwise, I don't know, it just seems to me that everything else would just fit in nicely - no DNA left to test, rusted clasp, if the cops were that bad even the Lumumba episode could be forgiven.

Chris Halkides said...

Gallagher,

As you know from the previous thread, TomZ disputed Dr. Balding's assertion on that point, and he is a molecular biologist who does PCR. And as far as the Court of Cassation is concerned, they have made such nonsensical statements with respect to DNA that nothing they say in the future could surprise me. As far as I am concerned, neither the knife nor the clasp should have made it into the courtroom, on multiple grounds. I would say the same thing even if I thought that the pair were guilty, which I obviously do not. And it would have been better for Italy in the long run had this happened. The police need to lose one or more cases before they quit being so sloppy.

Purely as a matter of tactics, I don't buy into your argument. All that would happen is that the people making the accusation would get hit with charges of defamation or worse, and there is no reason to believe that the Court of Cassation would be any more receptive to this argument than any other.

Gallagher said...

Chris,

I know we disagree a lot, but I feel strongly that in the long run this can only be good for Italy. This kind of stuff all has to be brought out into the open and aired. If the Italian justice system is so flawed, this could be the spark for reform that's needed. The downside is that two people, who might be innocent get convicted, and there's no easy answer to that one.

On contamination, that's always bothered me for reasons I've already expressed, and I would probably rather go with guilt than accept it - just seems too far fetched. I was watching extracts from the CNN documentary, they did a pretty good job from their perspective, over the top but OK. But the bit towards the end, after everything's been wrapped up, and they ask the fat guy who's commenting - "so what happened to the clasp", and he simply shrugs his shoulders and replies - "contaminated", and I just thought - what! after all that you're bound to be able to come out with something better.

Anonymous said...

Two fabulously interesting articles:

http://www.abajournal.com/magazine/article/crime_labs_under_the_microsco
pe_after_a_string_of_shoddy_suspect_and_fraudu/?utm_medium=email&utm_cam
paign=weekly_email&utm_source=maestro&sc_cid=130904BJ

http://www.nytimes.com/2013/09/06/nyregion/as-doubts-over-detective-grew-prosecutors-also-made-missteps.html?hpw&_r=0

Enjoy,
Observer

PS to Gallagher, the whole point of the people who believe knox and sollecito entirely, 100% innocent is that there is a standard of proof required to overcome the presumption of innocence. The "proof" in this case has red flags all over it. Even so and despite the fact the bra clasp and the knife were collected under extremely odd circumstances and provided utterly unreliable and inconclusive evidence, that should be the end of it...the evidence should not have been permitted in court. As you so vividly demonstrate, lots and lots of people want to believe prosecutors, regardless of how poor the quality of the evidence or how ridiculous the theory of the case. But, regardless of the havoc and hell the prosecutor and his team wreaked in this case, the requirement that solid evidence is required to demonstrate guilt carries over, even to the benefit of the prosecutor's team. The simplest and most likely explanation for the DNA fiasco is contamination. If DNA evidence can be contaminated at all (and it surely can!!), there is every likelihood that the evidence was contaminated based upon both the collection methodologies and the quality of the DNA collected. There is no burden of proof on the defendants (at least theoretically and by law) to prove that someone tampered with the evidence. So, until and unless there is solid evidence someone tampered with the evidence, why make that claim? I think that's the mantra of the pro-innocence group...and, frankly, isn't it the bedrock (at least theoretically and by law) of our criminal justice system? A suggestion: read "Until Proven Innocent" by KC Johnson and the story of Cameron Todd Willingham (http://www.newyorker.com/reporting/2009/09/07/090907fa_fact_grann). These two factual accounts explain graphically how every single institution we revere (elite colleges, highly ranked hospitals, prosecutors, detectives, crime labs, science experts, clergy, elected officials) are capable of working together almost unconsciously and synergistically to achieve a completely incorrect and unjust outcome. That's why the proof just has to be solid. And well meaning people like you are why grossly weak evidence should never, ever, ever, ever be permitted in a court of law.

Gallagher said...

Chris,

It may have come to your attention that you have an anonymous poster/blogger wishing to mark closure on commentary. Very softly spoken and gentlemanly, so I think we can write off Sfarzo. Maybe it's you, maybe it's Kaosium without the flatulence, don't know, but I like it. Kind of reminds you of the epilogue after an old Hitchcock movie with the curtains coming down. Anyway, thanks for your hospitality and I enjoy my time here. Catch you again.

Chris Halkides said...

Gallagher, I have no idea what you are talking about.

Chris Halkides said...

Gallagher,

You wrote, "I know we disagree a lot, but I feel strongly that in the long run this can only be good for Italy. This kind of stuff all has to be brought out into the open and aired. If the Italian justice system is so flawed, this could be the spark for reform that's needed. The downside is that two people, who might be innocent get convicted, and there's no easy answer to that one."

I strongly agree that the problems of this case go beyond the fates of two innocent people who may yet be convicted. I also am not the only person who got involved partially to effect change. No country has figured out how to handle forensic evidence in a courtroom perfectly, but Italy is needlessly behind the curve in certain matters. Nothing would please me more than to see it catch up with and then pass other nations, or at least force them to keep tweaking their systems to keep pace.