October 5, 2009

Seven Deadly Sins of the Knox/Sollecito Prosecution

“Sin creates an inclination to sin; it engenders vice by repetition of the same acts.”

First Posted October 1, 2009
We've talked about the scientific "trees," DNA, Luminol, and the rest. Now let's take a step back and look at the forest of the Knox/Sollecito prosecution. Although science can exist in a perfect void of human issues, the science in this case is shrouded in a forest of human intrigue that cannot be ignored. A strange, dark, and twisted forest, it is.

The ordeal of prison and trial has gone on for nearly two years. Yet the real decision was made just a few days after the murder of Meredith Kercher. Head investigator Edgardo Giobbi determined, in his mind, the guilt of Amanda Knox and Raffaele Sollecito before he had even begun to acquire, let alone analyze any actual evidence.

He boasted of the feat on film. “We were able to establish guilt by closely observing the suspect's psychological and behavioral reactions during the interrogations. We don't need to rely on other kinds of investigation as this method has enabled us to get to the guilty parties in a very quick time." This was achieved before he even heard of Rudy Guede. In fact, just hours after the murder, when he handed Amanda a pair of shoe covers, "As she put them on she swiveled her hips... my suspicions against her were raised.”

Of course, these same investigators were confused by the meaning of Amanda's enigmatic text message to Patrick Lumumba, “See you later.” They thought it meant something like, “Let's meet later tonight and commit a senseless, horrific crime.” But despite this cluelessness, or perhaps because of it, deep psychological insight enabled them to perceive guilt in the complete absence of evidence. It might be called, a “thought crime."

That is why, nearly two years later, even though there is still no evidence against Amanda and Raffaele, these investigators continue to fight for convictions. Their minds are made up, and they are not to be confused by mere facts.
That was the original Sin of the this murder investigation. The Sin of Pride, excessive belief in one's own abilities.

Next came the theory of the crime, a compelling story in which to wrap the guilt. This naturally revolved around a satanic sex orgy gone bad. Naturally to whom? To the prosecutor, Giuliano Mignini. There was no evidence of this. No prior history, no such discussions, no records. In fact, in the entire trial there has never been any testimony about this, nothing ever offered by the prosecution. Just leaks to the press, who continue to parrot phrases like, “sex orgy gone bad.” And just young people who, shockingly, have had sex . And one was an American. You know how they are, and a beautiful one at that.

All of that, and a coincidence of date, the day after Halloween, along with a helpful suggestion from a blogger who channels a dead person to reveal a Satan worshipping Masonic sex cult (I'm not making this up) was enough for Mignini. In his pious mind, he envisioned the same lurid evil he had confronted before, in the Monster of Florence case. With much the same result, another tremendous injustice, perpetrated by Mignini himself.

That was the second deadly Sin of this investigation. The sin of Lust, an unnatural obsession with carnal desire.

In the interrogation of Amanda Knox, the result was preordained, the only question was how long would it take, how much pressure it would require, to put the words into her mouth to seal her fate. No matter how many times Amanda said she didn't know, that she had no idea who had done this thing, they told her that she did, and that she'd spend 30 years in prison if she didn't say what they wanted her to say. Day after day they brought her in. Forty some hours without a lawyer, without a good night's sleep, without an interpreter, interrogating her in a language she barely knew. How long would you hold up?

They asked her to envision the murder, asked her to picture it in her mind. They brought Patrick into it. Why, she had sent him that incriminating text message, “See you later.” They told her that Raffaele no longer supported her when she said that she was with him that night. Suspects must always tell the truth, over and over again, without fail or variation. Interrogators are under no such restrictions.

And so she broke, and told them what they were demanding to hear. As soon as she possibly could, she wrote that while she had said such things, they weren't to be relied upon, “But I've said this many times so as to make myself clear: these things seem unreal to me, like a dream...” But, for the prosecutors, hey, close enough. They had her cold. The press forever after has called this her “confession” or “false confession.” More on the press in a future article.

What she really said in the middle of the night, what really happened, what coercion occurred, we may never know, because the prosecution will not release the recordings.

And so they committed the third Sin, Wrath, the emotion of the falsely righteous” “which can manifest as vehement denial of the truth.”

Witholding evidence from the defense has been a powerful, ongoing tactic of the prosecution. You can't refute the data if you can't analyze it. You can't analyze the data if you don't have it. The prosecution was finally forced by the judges to release information they had previously withheld on July 30, 2009, more than a year and a half after it was gathered. But their hoarding continues. Several critical pieces of information remain under wraps. These include:

The recording of Amanda's interrogation. The Polizia recorded everything. Phone calls, conversations, chats. It is not credible that Amanda's interrogation was not recorded. Yet the recordings have been conspicuous by their absence. This was either incompetence for not recording, incompetence for losing the recordings, or, far more likely, the prosecution is withholding evidence that looks bad for them.

The dates that DNA profiles were performed. The Knife (that doesn't match the wounds) was collected from Raffaele's kitchen drawer after most of the DNA containing evidence was gathered from the apartment. It is extremely probable then, that Meredith's blood and DNA were analyzed in the days immediately before the knife DNA testing. If so, that would provide strong additional evidence that the low copy number knife result was from contamination. The Knife would have been tested in a lab environment suffused with Meredith's DNA. Those dates remain hoarded by Stefanoni and the prosecution.

Results of blood tests from Luminol reactive sites. Luminol glows are only “presumptive” of blood, and are certainly not conclusive. The next step when a glow is seen is to swab the area and use a test that is specific for blood. It was never credible that these simple tests were not done. In fact, they were performed, but were withheld, apparently because they didn't look good for the prosecution. As Sara Gino stated "We were not told that, first of all, the prints were treated with a substance which should have indicated whether they were blood, and the result was very uncertain."

These, and other acts comprise the fourth Sin, Greed, or Hoarding of materials or objects, by means of trickery, or manipulation of authority.

In my last article, Methods of the Polizia PseudoScientifica, I made the statement, “These people mostly know what they are doing.” That was an exaggeration, for which I apologize. In fact, if you consider the drumbeat of incompetent performance they have compiled, it is clear that they only rarely know what they are doing. And those rare times are mostly when they are distorting the evidence.

1. There have been many failures in the forensic effort. Three out of four critically important computer hard drives were destroyed by the Polizei “computer experts,” Amanda's, Meredith's, and one of Raffaele's. The remaining critical computer drive, Raffaele's laptop, was merely altered by being used by the police to surf the net, destroying evidence that could have proved Raffaele and Amanda's alibi.

2. When a murder victim is found a, measurement of body temperature and room temperature is a fundamental procedure as a means of indicating time of death. These measurements need to be taken as soon as possible for the most accurate information. Every competent investigator knows this. Meredith's temperature was not measured until 1:00 AM the day after her body was found, more than 48 hours after her death and far too long to provide meaningful information.

From Meo Ponte, La Repubblica
Professor Giancarlo Umani Ronchi:"We were asked to determine time of death," he explains, "but they had already made outrageous mistakes." "First, the failure to analyze the corpse by the coroner who arrived first at the scene."

3. A surveillance camera at a bank across the street from the apartment recorded all activity on the street, and would have seen the perpetrator coming and going. Unfortunately, the Polizei didn't get around to accessing it until the recordings had been overwritten.

By these and many other actions, the Polizei have committed the Sin of Sloth, the avoidance of work.

Patrizia Stefanoni had neither the proper equipment nor the proper laboratory to perform low copy number DNA profiling, but she did it anyway. There are only a few such laboratories in the world, as it is a very new, as yet unproven technique. Her own lab was not even certified to perform ordinary DNA profiling at the time these tests were performed. The result was that she performed tests that do not conform to any standard, any where. Now she is withholding the information about when and how those tests were performed.

And so she committed the Sin of Envy, the perception that someone else has something they are lacking.

What lies behind this prosecution? Some have said that Mignini actually thinks that Amanda and Raffaele are guilty. But to believe that it is so simple would be naive. Italy is the home of an idea called “dietrologia” “the science of what lies behind” that involves seeing the real truth behind the obvious facade. Preston and Spezi give a great background on this in Monster of Florence. “Dietrologia is the idea that the obvious thing cannot be the truth.”

And what is this dietrologist's explanation of the motivation behind the prosecution?

It is Gluttony, in the form of relentless pursuit of money, position, and power. “The chief error about gluttony is to think it only pertains to food.” Gluttony is an inordinate desire to consume more than that which one requires.

There are careers to be made, and careers that could be broken, in such an infamous case. There are multi-million dollar lawsuits, all hanging on a guilty verdict. Rudy, you see, has no money.

And so we have a feast of innocents, gorged upon by a circle of gluttons. It is a large, and festive circle, with Mignini at it's head. There is Patrick, hoping for his half million, and Maresca, who would bring his clients tens of millions, wanting for his cut. There are those hoping for career enhancement; Giobbi, Stefanoni, Commodi and the rest. And even the ordinary polizei, who drove the unlucky pair to prison for the first time, just days after the crime, with their car horns blaring in victory.

The gluttons have only to commit two young innocents to life in prison and they will have what they desire. They have tasted fame, and they desire more. They have money, but they want more. They have positions of power, but one can never have enough power.

Gluttons are never the ones to say, “Enough!” That word must come from higher powers. From the judge and jurors? From the Italian government? The Italian people, or press? Or, perhaps, from a power, higher still?

Video by Andrew Lowery Anderson promoting the site, http://amandaknox.it with my articles posted in Italian.

Methods of the Polizia PseudoScientifica: A Knife, a Clasp, a Glow

First Posted August 28, 2009

We have had time to consider the methods of the Polizia Scientifica in the Knox/Sollecito trial, and they have come to look, not merely unscientific, but pseudoscientific. That means pretend science, it is not the real thing. We see this, because a consistent pattern has emerged.

It is not a pattern of innocent mistakes, made under the stress of time. Neither is it one of incompetence, for these people mostly know what they are doing. Neither does it appear to be blatant framing by splashing blood, planting evidence, or manufacturing data. That type of lie would produce clear results, and might also leave clear tracks. These results are anything but clear. And who wants to leave tracks? Certainly not the Polizia Scientifica.

What appears to be the pattern in the Knox/Sollecito trial is something like this: The Polizia Scientifica plucks an item from the field. They subject it to of some sort of “scientific” testing. Then they “cherry pick” the results, and present these special results in the complete absence of anything to compare them with. That is, without any control experiments. This modus operandi is compatible with pseudoscience. It appears to be an attempt to create the appearance of scientific certainty, while producing absolutely unscientific results. Who are they trying to fool? The judge and jury.

“...pseudoscience is any subject that appears superficially to be scientific, or whose proponents state that it is scientific, but which nevertheless contravenes the testability requirement...” http://en.wikipedia.org/wiki/Pseudoscience

What is this “testability requirement?” It means that if you can't test it, if you can't repeat the experiment to see if it happens again, it isn't science. It is pseudoscience. Take the kitchen knife DNA. The testing performed by Stefanoni destroyed the sample. Not a trace was left. Her experiment cannot be tested, cannot be reproduced. It can never be checked to see if it was a real result, or a fraction of a flyspeck of contamination from her lab. It was, “I did this experiment. These are the results. Trust me.”

Here's the procedure in more detail. Follow along. There will be a quiz at the end.

Step 1: Collect some stuff from the crime scene, or thereabouts. A kitchen knife, a clasp, a Luminol glow. No need to be picky here. It's not the item itself, it's the science you do to it that makes it cry “guilty.” That's why it didn't matter that the kitchen knife was a randomly chosen implement, as shown in “The Magic Knife.” A can opener from Raffaele's drawer would probably have Amanda's DNA on the handle, it just wouldn't sound as impressive.

Step 2: Perform tests on the item. What kind of tests? Scientific Tests, of course. The tests need to have the veneer of impressive, and barely comprehensible science upon them. For this, DNA profiling cannot be beaten.

Step 3: “Cherry pick” the results. This means, pick the ones that support your theory, and leave the ones that don't. Don't like a result? No problem. Don't report it. Discard those DNA peaks that don't fit. Didn't find anything that incriminates the defendant? No problem. Go back 46 days later, and pick up some more stuff from the scene.

Step 4: Present these results floating freely in an ominous air of suspicion and guilt. Leave out any kind of reference, any kind of comparison that might show how uninformative and mundane they really are. Avoid, in other words, any kind of control tests.

Now you have the impressive sounding results to support the prosecutor's tales.

Let's talk some more about control tests, because this is such a fundamental matter, that it is often lost in the grass. An experiment without a control is like a thermometer without a scale. You see the mercury inside, but you have nothing to compare it with. You can understand that it is meaningless, it is data without context, or, you can point at it and make up any temperature you want.

“Scientific controls are a vital part of the scientific method, since they can eliminate or minimize unintended influences such as researcher bias.” http://en.wikipedia.org/wiki/Control_experiment

Careful scientists take steps to keep their own researcher bias from influencing results. They perform double blind experiments, for instance, in which even they don't know which specimen is which, to prevent themselves from knowing what the results “should be.” That way, when they get a result, they can trust it, because they know they didn't unconsciously choose it.

Let's walk through the Polizia PseudoScientifica process for three items.

A Knife
The kitchen knife was retrieved from Raffaele's kitchen drawer some 5 days after the murder. It was reportedly taken to the police station, sat on a detective's desk for a day or so, and then was mailed to the lab in an ordinary box. Hardly the kind of careful handling you would expect of evidence slated for conventional DNA testing, let alone the hypersensitive LCN profiling. The blade DNA profiling was performed by an improvised, non-reproducible, never-validated method with deficiencies described in detail in LCN DNA Parts I and II. In fact we can add extremely poor evidence handling to the list of 9 deficiencies compiled in Part II to make it 10 testing deficiencies.

The low copy number test on the kitchen knife was apparently performed without any negative controls. In negative control tests you perform the DNA multiplication and profiling, without adding any sample to the system. Often when this is done, as if by magic, a DNA profile emerges. It has come from a minute amount of contamination from the equipment, or the laboratory. These negative controls are essential to performance of LCN work, as shown in Part II of this series. They were either not performed, or not reported by Stefanoni.

Perhaps even more important for the knife DNA, no control experiments were run to follow the handling of the item from the field through to the laboratory. That is, to see if other, random objects retrieved from the same drawer and handled in the same, unprofessional way, might also appear to have DNA on them. It would be interesting to hear the prosecution spinning a sinister implication out of DNA found on a can opener. Perhaps one can use canned peas for satanic rituals. Would Meredith's DNA be found on a spoon from the same drawer? How about Filomena's? Would the spoon then be cast as the murder weapon, whether it matches any wounds or not?

All this is preposterous of course. But think about it. We have no way of knowing what the supposed knife DNA means, or where it came from, because no comparison tests of any kind were performed.

A Clasp
Making sense out of the bra clasp DNA, which reportedly includes profiles from approximately five people, would be a challenge even with careful, rigorous control experiments. Without them, it is hopeless. Ask yourself this simple question. What would happen to any random object left on the same floor and kicked about for 46 days? Especially an object with cloth attached , making it a virtual dust mop. It would be covered with dust, and the DNA that comes with that dust. Raffaele was at the apartment visiting Amanda on several occasions. The presence of his DNA there means nothing.

Control experiments to check for this would have been simple. The clasp was retrieved from a pile of debris left by the fastidious investigators in Meredith's room, shown in the picture at the right. Testing a few other items from that pile to see if they, too, had picked up DNA dust from the floor would tell us whether there was anything special about the clasp. Of course, that wasn't done.

Collection of the clasp by the Polizia Scientifica

So we have “Raffaele's DNA was found on Meredith's bra clasp,” rather than, “Raffaele's DNA, along with DNA from lots of other people, was found at various random locations throughout Amanda's apartment, which he visited several times before the murder.” The first phrase sounds incriminating. The second, accurate phrase, shows how meaningless this test result is without a control experiment.

The handling of the clasp when it was retrieved from the scene is shown in the video above. The investigators, dressed in fancy white outfits, seem to play some kind of game with it. Why the outfits? They do nothing to prevent mixing contamination of the material at the scene. As shown in the closeup picture, the outfits, and their gloves, quickly become contaminated by DNA from various sources at the scene which can then be transferred to the evidence.

A Glow
Luminol glowing footprints were found in a hallway, and some may have been Amanda's, it is hard to know for sure because they were only compared with her feet, and found to be “compatible.” Again, no controls. Meredith, Laura, Filomena, none of the other resident's feet were compared to these footprints. The footprints were tested for blood, and it came out negative. No blood. So, why are they important? Amanda lived there, after all.

Amanda's DNA was said to be found in one of these footprints. Did they also test a meter away from the footprints, to see if her DNA was all over the apartment where she lived? No. That would have been another control experiment. Was the DNA actually associated with the footprint, or did it just happen to be there, because the resident's DNA was all over their apartment, as people's DNA usually is? We will never know. They skipped the control experiments, and presented results without any reference.

It's getting hot in here. Just look at that thermometer without any scale!

These are just three examples, there are many more. Enough to discern a clear pattern, that the methods of the Polizia Scientifica are compatible with pseudoscience, and are a consistent attempt to mislead the judge and jurors.

Now for the quiz:
1. If you wanted to perform honest tests to search for truth, to learn what has really happened, would you exclude control experiments? (Yes /No).

2. If you wanted to perform tests that appear scientific, but are actually intended to make an innocent person look guilty, would you perform control experiments? (Yes/No)

3. Did the Polizia Scientifica perform control tests in the Knox/Sollecito investigation? (Yes/No)

If you answered “No, No, No!” then, you've got the point. If you didn't, stop and think if you would like to be investigated with these same kinds of tactics.

Special thanks to my molecular biologist friend, Dr. K. for reviewing these articles.

LCN DNA Profiling Part II: Watch Where You Sneeze

First Posted August, 16, 2009

When people's lives and liberty are on the line, you hope the people on the job get it right. That is part of what is so disturbing about the technical work of Dr. Patrizia Stefanoni of the Polizia Scientifica. When Amanda and Raffaele's liberty was at stake, Stefanoni “winged it.” Made it up as she went along. She performed science improv. However you put it, her techniques were at odds with both accepted scientific methodology and with simple common sense.

Some of the serious issues surrounding LCN DNA profiling, shortened here to “LCN,” were described in Part I of this article. Now we pose the question: How does the test performed by Stefanoni on the kitchen knife blade DNA compare with the techniques called low copy number DNA profiling?

In a nutshell, it was worse.

Here is the account given of Stefanoni's work by Frank Sfarzo in his Perugia-Shock blog at http://perugia-shock.blogspot.com/2009/05/lab-of-wonders.html

“But the substance was minimal and if she took it to test the blood than nothing would remain to test the DNA. So she said O la va o la spacca, make it or break it, and took a 20% of it to test it for blood: negative.

The test failed but Dr Patrizia wasn't discouraged and she took what remained, about 20 microliters, she dried it to 10 and tested it for DNA. It didn't sort anything but Mrs Stefanoni didn't give up and started to amplify and amplify until the first peaks appeared. The machine was not allowed to go beyond, but something there was and had to be taken out. The goodwill scientist broke the seals and kept amplifying and amplifying and amplifying until, in a forest of background noise peaks, some alleles emerged. She decided which were the stutters, the false ones, and which the real alleles, et voila! The genetic profile of Meredith Kercher was served to Renato Biondo who could deliver it on a silver plate to the one who hired him, the prosecutor.”

There are two common definitions of low copy number DNA profiling. The first states that profiling that falls below the normal stochastic limits can be considered LCN profiling. That is, when random noise becomes very loud because the sample size is very small, you have LCN, not standard DNA profiling. The second definition uses some quantity threshold criterion. If you have less than 200 picograms of DNA, for example, it is LCN. The critical factor is that the number of original template molecules in the sample is very few, on the order of 5 – 10.

The profiling that Stefanoni performed on the DNA from the blade of Raffaele's kitchen knife meets either of these definitions. There was so little DNA present that the instrument indicated no DNA until Stefanoni overrode the machine limits. This amplification increase was not achieved by the PCR technique. Once the sample has been chopped up and subjected to electrophoresis, it is too late for that. The increase was performed by other methods, such as lowering the threshold level, or simply changing the display scale until the minute fluorescence peaks were visible.

This makes the testing performed by Dr. Stefanoni even worse than the various methods being experimented with to perform normal LCN testing. In those cases, the experimenter knows that the sample is tiny, and tenuous, so they take appropriate precautions and use a higher number of PCR multiplication steps, typically 34 compared to 28, resulting in 2^6 (64) times as many molecules for the electrophoresis and fluorescence observation.

But remember that the main problems with LCN are introduced during the first few cycles of replication, when there are very few molecules and any difference in replication becomes a major artifact. That happened with the kitchen knife DNA, so all of those errors are well represented. In this case, however, not only were those artifacts introduced, but additional artifacts from the high amplification of the weak fluorescent signal also added noise to the vanishing weak signal. So that's one deficiency.

Yet another troubling question is raised by this mid-course change in experimental methodology. Since Stefanoni did not originally expect to perform LCN profiling, but only conventional profiling, she apparently did not observe any of the far more stringent protocols that are observed by other laboratories experimenting with use of LCN. These include extreme efforts to avoid contamination, provision of control tests, retention of a portion of the sample for subsequent testing, performance of the test on two samples for comparison, and clean up processing steps.

Even with those far more stringent protocols, LCN profiles are not allowed as evidence by the vast majority of the world's courts. But let's take a look at these proposed procedures that attempt to place LCN testing on a more reproducible, reliable basis. That way, we can illuminate the deficiencies in Stefanoni's science improv technique.

The following quotes are all from the Crown Prosecution Service in the U.K. at: http://www.cps.gov.uk/publications/prosecution/lcn_testing.html This is from an article that supports the use of LCN.

“The FSS LCN test requires an ultra-clean laboratory and so is more expensive and less widely offered than the standard test.... The site of this bespoke laboratory is remote from other DNA Units, operates stringent entry requirements, is fitted with positive air pressure and specialist lighting and chemical treatments to minimize DNA contamination.”

Stefanoni's procedure, in sharp contrast to these requirements, was performed in an ordinary DNA analysis laboratory with other DNA units present. An ultra-clean laboratory, positive air pressure systems, and photo and chemical DNA sterilization are vital techniques to avoid contamination of samples within the laboratory. None of these facilities and procedures appears to have existed for Stefanoni's test. That makes four more technique deficiencies.

“In LCN testing, each sample is divided into three parts or aliquots, and two of these are tested. The third is retained for further testing in the event of a failure or to confirm the presence of a mixture.... Only those DNA components that are seen twice are included in any calculation, to show that the result is reproducible.”

Stefanoni used 20% of the sample to test for blood, which came out negative. Whatever was on the knife, it wasn't Meredith's blood. Then she tested all of the remaining material at once, so there was no possibility of comparing two results, and nothing left for further testing. By the standards of the Crown Prosecution Services, Stefanoni's results would be thrown out by either of these criteria. And that makes two more deficiencies.

We're not through. Laboratories performing LCN rely heavily on what are called “negative controls.” The following quote is from The Law Society of Scotland's publication at: http://www.journalonline.co.uk/Magazine/52-2/1003857.aspx

“In forensic science the fact to be established is that the DNA profile originated from the material recovered from a crime scene or a suspect, not the investigator, the laboratory, packaging, or analytical instruments. A “negative control” is set up by simply processing a “blank” sample that has no DNA. All being well, this control will not show any DNA. The presence of DNA in the negative control illustrates that there has been a source of contamination in the analytical method. It does not, of itself, show where that occurred, merely that it has. The tradition over many years has been, for very sound reasons, that anything found in the “negative control” invalidates the analysis.

...even in a tightly controlled analytical procedure a significant number of supposedly negative controls give a positive result, i.e. they indicate the presence of DNA.”

Stefanoni apparently did not perform any negative controls with same system parameters as that used for the kitchen knife DNA. That's another deficiency.

And note here, the statement, “It does not, of itself, show where that (contamination) occurred, merely that it has.” When Sara Gino was pressed, under cross-examination, to say where and how Stefanoni contaminated the samples, she could not give a specific answer, but only cited, “the literature.” This was seen as a weak response. But how could she possibly know at what point in the handling or processing contamination by 50 picograms of material may have happened? This is something you test for the presence of, not something you can possibly see happen. Even if someone followed the sample with a microscope in real time, it would be virtually impossible to witness the inadvertent transfer of such a minute amount of material. Gino's response, that she could "cite the literature,” which lists typical sources of contamination to guard against, was correct.

But these negative controls do not check for all sources of contamination, they only check for contamination originating in the laboratory. If the kitchen knife were contaminated before being chosen at random from Raffaele's drawer, this type of control won't catch it. If it was contaminated while being picked up, while being transported, or anywhere else along it's path, this method will not show that. To do that, you would have to perform additional negative controls, say, on the other knives in the drawer, or from some other source, handled in the same manner as the kitchen knife. None of this was done. That's yet another, major deficiency.

So we see that in roughly nine distinct ways, Stefanoni's improv LCN DNA profiling was even worse than unproven and inadmissible LCN DNA profiling tests.
1. The DNA wasn't amplified enough; the very weak fluorescence was simply blown up.
2. The test site was not remote from other DNA tests to avoid contamination.
3. Specialized LCN-quality entry procedures to avoid contamination were not used.
4. A positive pressure environment was not maintained to exclude contamination.
5. Special LCN sterilization procedures to destroy errant DNA were not used.
6. The entire sample was consumed in a single test; no comparison of tests was possible.
7. No sample was retained for future reference. The test can never be reproduced.
8. No negative control tests were run to check for contamination.
9. No control tests to check for field contamination were performed.

There could be some mistakes in this analysis. Unlike Stefanoni, who claims that there has never been contamination in her laboratory, I make mistakes. It may be six deficiencies, or an even dozen. We do not know the exact details of how samples were handled in the laboratory of Patrizia Stefanoni. Neither does the defense team, or the court, as determined by Judge Massei when he ruled that she needed to provide more information. In fact, when you see the clear discrepancies between what was said to have happened, and what you can see happening on the specimen collection video tapes, it is clear that not even Stefanoni really knows what transpired during the collection, transportation, storage, and subsequent analysis of samples in this case.

There are, perhaps, some things that she should have known, that clearly went wrong. A glove not changed, as evidenced by the crease in the exact same spot after she claimed to have changed it in between samples. A specimen handled by more than one person, another transported in packaging that was not sterile. A specimen retrieved, and then dropped on the floor, deliberately or otherwise, after it had been on that floor for 47 days after the crime.

But, perhaps most importantly, in this realm of sensitivity, in this extreme, almost ethereal realm where picograms determine everything, it is just not possible for ordinary handling measures to prevent contamination of the results. The DNA on the kitchen knife blade is more likely to have been contamination, than to have been a valid forensic sample.

Special thanks to my molecular biologist friend, Dr. K., for reviewing these articles.

Canary in the LCN DNA Mine, Part I

August 7, 2009

It's called “Low Copy Number” DNA profiling (LCN DNA), and it could be a powerful tool against crime, or a chilling assault on our freedom. It is the technique used to claim that the kitchen knife found in Raffaele Solecito's kitchen drawer had Meredith Kercher's DNA on the blade. If it is used to convict Amanda Knox of murder, she may well prove to have been the “canary in the mine,” drawing our attention to a potentially serious threat to civil liberties everywhere.

We have all heard about the remarkable abilities of DNA profiling to identify criminals and others from traces of their blood or bits of their skin. We have been told that the odds of a mistake being made are “billions and billions” to one in Carl Saganesque tones of certainty. But this new twist on the technique radically changes those odds, and not in a good way.

Conventional DNA testing is done with a microscopic, but still significant size sample of DNA, on the order of 1 nanogram (1 billionth of a gram). This quantity provides enough material to ensure that it is physically associated with the actual evidence at a crime scene; a smear of blood, a patch of hair, a cigarette butt. You extract a sample from the specimen, and profile it. And you still have the specimen. You can extract a second sample, and test it again. Or pass it to the defense for their comparative analysis. The experiment is reproducible because there is enough material present to do the test more than once.

“Interpretation of DNA profiles is assisted by the use of   systems that are not too sensitive. This is important   because the scientist often needs to associate the presence of a bloodstain (or other evidence) with the DNA profile itself.” Peter Gill, Forensic Science Service, U.K. Article available online at: http://www.denverda.org/DNA_Documents/LCN%20DNA%20Profiling.pdf

This DNA is replicated by a method called the Polymerase Chain Reaction (PCR). This reaction splits the two halves of DNA apart, adds complementary base pairs to each of the halves, and voila , you have two DNA chains where before you had one. Do it again, and you have 4, 8, 16, 32... 268 million (2^28).   Ideally, each cycle of PCR gives twice the number of copies, so you have 2^N copies after N replication cycles.

It is then diced up in a selective, specific way, and the pieces are spread out by a method called electrophoresis, which drives different lengths of DNA at different speeds through a gelatine material. The pattern of these different lengths is then analyzed statistically, and compared with the known DNA from various people. If the patterns match, the DNA is thought to match.

This much is straightforward. Conventional DNA profiling is supported by a well-established body of scientifically controlled experiments, it yields reproducible results, and these results have been extremely valuable, both in identifying the guilty, and in exonerating the innocent. But LCN DNA testing is a different matter entirely.

Low copy number DNA profiling is usually performed by increasing the number of replication steps compared with conventional DNA testing, so that an even smaller sample is blown up to a large enough quantity for electrophoresis. Every added step roughly doubles the number of DNA chains, but also introduces exponentially greater chances for errors in the replication process. This is particularly important when the initial sample is very small.

When enough DNA is available as the starting “template,” the copying process is performed on a large number of molecules right from the start. Any small variation in whether or not a given molecule copies, and what parts of it copy, are washed out because there are a lot of templates available. If you miss one in one hundred, you only have a 1% error. However, if you only have a few template molecules, and any part is missed in the first stage, that error continues to be amplified throughout the PCR process. Miss one in five, and the error for that single step jumps to 20%. Some profile peaks may be diminished, some may be increased, some may drop out entirely, and “stutters” may occur, poorly understood false peaks that are, in a sense, combinations of other peaks.

The statistical variation resulting from the tiny number of starting templates is significant. In fact, LCN DNA profiling is usually defined as either testing with a very small amount of starting material, say 100 picograms, or as profiling with results that fall below the normal stochastic limits of the technique. “Stochastic” means that an element of chance is involved, so that the system is not deterministic. It contains a significant amount of random noise. As a result, repeating the same LCN DNA tests on identical starting samples of material does not produce nearly identical profiles, unlike conventional DNA testing.

The tests performed on the knife DNA by Stefanoni should be classified as LCN DNA profiling by either of the two definitions. The amount of material appears to have been well under 100 picograms, and the test results from that were below these stochastic limits. The same random fluctuations that plague LCN DNA affected her test results, because the number of starting templates was extremely small. She did not, however, use additional PCR cycles to increase the amount of sample present. More on that, and other discrepancies in Part II of this paper, which will show that her techniques were significantly inferior to the emerging LCN technique.

Stefanoni's technique shares another problem with LCN. If the sample size is small enough, there is nothing left after the replication and analysis to repeat the experiment. It cannot be reproduced. In scientific work, an irreproducible result is automatically suspect because there is no way of confirming it. There is even a famous science humor publication named the Journal of Irreproducible Results (www.jir.com).  

LCN DNA profiling can be an irreproducible technique for two distinct reasons. First, because the sample is too small for conventional testing in the first place, it is usually consumed and destroyed in the course of performing the LCN test. No sample, no reproducibility. Second, the test results that are produced contain this strong, random variability. If you perform LCN DNA profiling on ten identical samples, you can get ten different profiles, each differing from the others because of amplification of statistical flukes.  

In addition to the issue of reproducibility, LCN DNA is simply a very new technique, and that should prompt caution. In fact, most researchers in the field fill their papers with appropriate caveats about the need for further development, and further testing before the technique is widely used. Conventional DNA profiling has been rigorously tested, reproducibly performed in many laboratories around the world, and validated with carefully designed control tests. With LCN DNA profiling, no such body of controlled tests yet exists. The method is too new, too quirky, and too unreliable to be safely used until it is much better understood.

Now let's think about what all this means for civil liberties. How would you like to live in a world in which any person can be convicted of any crime, anywhere, any time, on the basis of unassailable, “scientific” evidence? The evidence will be unassailable, because there will be nothing left of it by the time the analysis is through. They will be able to swab an object at a crime scene, LCN DNA profile it, and present it in court with no risk of contradiction. It will be their word against yours, and they will have a bunch of apparent, “scientific proof” backing them up. This is a recipe for a police state.

What does it mean for Amanda Knox and Raffaele Solecito? The DNA profile from the knife blade in Raffaele's kitchen drawer that supposedly matches that of Meredith Kercher was performed by an inferior version of LCN DNA testing. It can never be reproduced. The “any crime, anywhere, any time” danger expressed in the previous paragraph is not hypothetical, it is actually happening to Amanda and Raffaele. This form of evidence amounts to, simply, “The defendant is guilty because we say so.” Yet it is even worse. It is an un-testable assertion backed up by a bunch of impressive charts and statistics and the magic words, “science” and “DNA.” It has all of the appearance of scientific certainty with none of the substance. It is not scientific, and it is anything but certain.

Keep in mind that there is nothing especially “Italian” about this frightening new investigative technique. Most of the early work was done in Britain, and it is being studied in forensic labs around the world. Be patient, LCN DNA matching is coming soon to a forensic laboratory near you. How can we be sure that the technique won't be grossly misused, leading to the convictions of many innocent people, not just these two? We can't.

While this article gives some background on DNA, a more thorough overall primer is available at: http://www.scientific.org/tutorials/articles/riley/riley.html

“Because LCN analysis by its nature is not reproducible, it cannot be considered as robust as that associated with conventional DNA typing.”

From: http://www.denverda.org/DNA_Documents/CMJ%20Budowle.pdf

“The ability of small amounts of DNA to produce false and misleading results is well-known and well-documented within the research community, where the technology originated.”

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