Knox/Sollectio: was the knife contaminated?

The Qubit Fluorometer

The Qubit Fluorometer, used in DNA analysis relating to the alleged murder weapon

One of the two exhibits in the trial of Amanda Knox and Raffaele Sollecito to be looked at by Stefano Conti and Carla Vecchiotti as part of their review of DNA evidence in the case is a knife, recovered from Sollecito’s flat and alleged by prosecutors to have been used by Knox to stab Meredith Kercher in the neck.

Conti and Vecchotti agree with the opinion of the chief police scientist, Patrizia Stefanoni, that Knox’s DNA was present on the handle of the knife and they don’t dispute the match between the DNA found on the blade and that of the victim. But they raise clear concerns that the DNA profile observed in relation to the blade could have been the result of contamination.

The reasoning behind this turns on their characterisation of the the DNA sample from the blade as “Low Copy Number” (LCN), essentially meaning that there were so few examples of the DNA in question within the sample that, according to mainstream scientific opinion, special measures would have been required so as to ensure the integrity of the testing.

In her trial testimony, Stefanoni appeared to accept the proposition that the sample in question may indeed have been an LCN sample. It appears difficult to be absolutely sure about this, because there is no accurate record of the quantity of originating material (Stefanoni has said it was “in the order of a few hundred picograms (pg)”). Quantifications during a process known as qPCR, it seems, would have given a strong indication, but there is no record of these having been performed – perhaps because of the very fact that the sample was so small as to be out of the range of sensitivity offered by the equipment that would have been used.

However, the review highlights two reasons to suppose that the sample must have been of an LCN-type quantity. Firstly, there are what are known as “peak imbalances” in the graphical representations of the DNA which were produced. In most graphical printouts of DNA tests, peaks come in pairs. In an imaginary perfect test, the pairs would all be of equal height to one another. That is rarely the case in practice, so a ratio of 3:5 is normally considered acceptable. But, it can be seen from the graphs reproduced from page 68 on in the review that some of the pairings are more imbalanced than that. Although these imbalances can be produced by a number of means, they can be indicative of the presence of LCN material, so this provides some support for the notion that we are dealing with LCN. Moreover, if you accept that the raw material was “a few hundred picograms” and note that the volume of the solution from which the DNA reading was obtained was 10 microlitres (μl), then it follows that the concentration of that solutions must have been a few tens of pg/μl. According to the review, 200 pg/μl is generally considered to be a threshold below which material should be treated as LCN.

One difficulty with analysing an LCN sample is that, because its presence in a solution is so faint, amplification is required during the PCR process (indeed, it seems clear that Stefanoni did carry out amplification on the sample). Think about this as being like turning up the volume on a cassette player to hear something that has been recorded at a very low volume. Inevitably you are also turning up the hiss. Background noise will normally be easy enough to distinguish from the genuine data, but the real problem is that, as you amplify, you may be amplifying faint traces of contamination that are also present. So, in our case, if there is a reasonable risk that Meredith Kercher’s DNA might have been present as a trace-level contaminant, then the test conducted ought not to be considered reliable.

Conti and Vecchiotti portray this a real risk that could have arisen at any stage of the investigative process, from the recovery of the knife through to the final tests in the lab. They point to a body of scientific literature recommended specific procedures, above what may typically be implemented, in order to minimise the risk of contamination in the recovery, handling and analysis of the evidence, and question whether such procedures were applied in the Kercher case. It is even suggested that the sample from the blade should have been tested in an entirely different laboratory.

I think the strength of this argument varies according to the context you are talking about. Failure to protect against contamination is not necessarily to be excused at any stage. But, from the point-of-view of the results we are considering, it only really matters if there is an appreciable risk that the data observed could have resulted from contamination. So, it should be considered that the location from which the knife was recovered – Sollecito’s flat – is not one where the victim’s DNA would be expected to be present, because it is somewhere where she had never set foot. Regardless of how suitable the procedures carried out there were, there does not seem to be a real risk of contamination.

Similarly, in between the flat and the lab, the knife does not appear to have been in any location where Kercher’s DNA might be supposed to have been present nor handled by anyone who had been in contact with Kercher’s DNA.

As a side note, the review suggests that secondary contamination can never be ruled out in DNA testing, points to an authority that detecting this type of contamination is more likely where LCN is concerned. For example, perhaps Sollecito shook hands with Kercher at some point, then carried her DNA back to his flat and contaminated the knife. In theory, it is probably correct to say that this possibility cannot be ruled out. But the prosecution may be expected to argue that this type of contamination is an ever-present and merely theoretical risk. What the court ought to want to ascertain is exactly how likely such contamination is.

Even if it might be argued that contamination from Kercher’s DNA at Sollecito’s flat or during the initial handling of the evidence is a remote possibility, it is certain that her DNA was present in the lab. At least 50 samples containing it were tested there, 20 or more of them prior to the testing of the knife.

For this reason, a key question for the court is whether the procedures in the lab were adequate for minimising the risk of cross-contamination of samples by the people working there, particularly given the apparent likelihood that the material in question was an LCN sample. The authors of the review don’t categorically say that they weren’t, but that they don’t have enough information to make a judgement. The court at the original Knox/Sollecito trial was satisfied that the standards of the laboratory were adequate in this respect. Will the appeal court take the same view? Will it feel obliged to hold the police scientists to higher standards in light of the review?

If the procedures in place (an actually implemented) in the lab are found to satisfactory, that is not necessarily all there is to it. There are checks on contamination that can be carried out but were not in this case, perhaps in part owing to the small quantity of material being studied.

Ideally, as was conceded by Stefanoni in the original trial, the sample should have been broken into two or more portions to be tested separately. Assuming that similar results would have been replicated on at least two occasions, this would have shown contamination to be less likely.

As a further precaution, the presence of some type of organic material might have been established. Now, it may be felt that the victim’s DNA is the victims DNA. What does it matter whether it came from blood, or skin or whatever? However, if the presence of some particular type of cell were known, then this would provide an indication against the idea that the results were contamination. Stefanoni seems to have performed a test for blood which was negative, and then proceeded on the basis that the sample was too small for the test to have worked properly.

It seems likely that dividing the sample simply wasn’t possible and, although Stefanoni may now wish that she had been more thorough in determining the biological nature of the material she was dealing with, that lapse is probably not enough in itself to invalidate her results.

A bigger problem to my understanding (although the review doesn’t stress it in the way I might have expected) is the issue of how control tests were performed. Standard procedure is to run negative controls, where the test is performed on a saline solution or something else not expected to contain any DNA, so as to check for the presence of contamination already present in the equipment or tube. A positive control should also be run, on a sample whose properties are known, in order to check that the equipment is behaving as it would be expected to (a bit like checking your bathroom scales using a bag of sugar).

These control tests should really be performed not only at the start of testing for each sample, but also when an amplification is performed on a sample. This is particularly important for the negative control, because of the possibility that the amplification will reveal contamination that may not be evident prior to the amplification. But Conti and Vecchiotti report that they see no evidence that this was done. Perhaps the bench will be satisfied if Stefanoni performed the controls without saving them electronically and is able to confirm that no problems were observed. But there’s a clear problem here for the prosecution if it emerges that the controls were not performed.

The most important things for discussion at the next hearing on 25 July will, I think, be the quality of the procedures in place in the police lab for reducing the risk of cross-contamination between samples and the issue of whether or not adequate controls were performed on the knife-blade sample. The reliability of the knife as a piece of evidence in the case really relies on those two things going in the favour of the prosecution.

10 Responses to Knox/Sollectio: was the knife contaminated?

  1. Bobbery says:

    Thanks, that’s a really helpful summary.

    Something tells me this is a big boost for the prosecution, though.

  2. katody says:

    I think quite important finding that you overlooked is that there was no DNA ( and no blood) in the crevices between blade and handle of the knife. Why Stefanoni failed to test it is another question about her professionalism. Anyway the experts did it and found nothing. They made it the first point of their conclusions and in fact it heavily implies that Stefanoni’s result is not connected with the crime.
    They also point out that no human cells at all were detected on the knife – neither by them nor by Stefanoni. It increases even more the probability that alleles detected came from the lab contamination, not from the specimen itself.

    The experts stress the possibility of secondary transfer that I think you underestimate.
    First – the knife were handled by multiple individuals that have been to the crime scene before, even on the same day. Their lacking approach in handling crime scene is noted, documented both on video and in testimony.
    Second – both Amanda and Raffaele frequented the cottage and his flat for more then a week. Both of them spent time in the cottage the day after the murder, touching surfaces that clearly had Meredith’s DNA on, without any protection. Amanda were taken to the crime scene another day and given only shoe covers. I don’t think Meredith’s DNA in the LCN amounts is unexpected in Raffaele’s flat.

    Concerning the controls:
    The experts are quite clear that there is no trace of negative or positive controls in the documentation that Stefanoni provided.

    • maundy says:

      I think you’re reading quite a lot that isn’t there, Katody. The failure of the experts to find blood specifically at the handle end of the blade doesn’t really “heavily imply” anything and the review doesn’t make any implication about that in its conclusion.

      I also don’t see where Conti and Vecchiotti stress the possibility of secondary transfer. They only seem to give it a passing mention in the abstract. On the facts you cite, only one person involved in the search at Sollecito’s flat – Dr Marco Chiacchiera – had been to the crime scene, and there was a four-day gap. He doesn’t seem to have ever touched the knife.

      As I say above, I think it’s likely that the court will want to know more in practical terms about the risk of secondary transfer. I don’t pretend to know what they will conclude, but it doesn’t seem obvious to me that there is a significant risk taking into account the actual facts.

      There is also a general theoretical risk of secondary contamination from Knox and Sollecito, I agree. But I think the defence will want to steer clear of arguing that this is increased because of their presence at the crime scene. Although what you are saying is fair enough, when you say that they had been “touching surfaces that clearly had Meredith’s DNA on”, what you are effectively saying is that they might have had the victim’s blood, quite literally, on their hands.

      With respect to controls, yes, the review is quite clear that there is no record of any controls, and that could be bad for the prosecution. I do agree that it is possible that there were no controls but, equally, they could have been done without any electronic record being kept. This would not have been best practice, but it’s perfectly possible. I’ve seen it stated that the software associated with the AB7700 machine forces you to record everything, but that simply isn’t true. You save a session if you want to. The controls, if they were carried out, and the test proper would probably not have been done as a single session, because you have to turn the machine off to change samples.

  3. Ted kopple says:

    Thank you so much for your hard work and prudence. This the only place I’ve been able find that isn’t grotesquely offensive in it’s stupidity and childish aggression. The chance of finding anyone willing to talk reasonably about this case is literally one in a million. I don’t bother reading any other blogs about this case, this is the one and only source I need for information and intelligent, calm analysis. Thanks again.

  4. Maundy,

    In a newspaper article Professor Alexander Kekule pointed out that Amanda herself could have carried Meredith’s DNA to Raffaele’s flat, a possible route of secondary transfer. Movement of DNA from object A then to object B is secondary transfer in this case but not contamination. By definition contamination can only occur once the knife is under the control of law enforcement. Officer Gubbiotti, the second officer to have control of the knife, had been to the girls’ flat that day, a possible route of contamination outside of the laboratory. However, the routes of contamination inside the lab must also be considered. Given that this sample falls into the low template number range (calling this sample “LCN” range would not be good terminology) and that none of special precautions of LCN testing were used, this is the most likely mode of contamination, IMO.

    The lack of blood is a serious problem for the prosecution, as I see it. According to Johnson and Hampikian if one cleans a bloody knife to the point where blood is no longer detectable, then it is unlikely that DNA remains. To believe otherwise one must invoke some kind of differentially effective cleaning, and I don’t see how that is possible. The presence of starch on the blade suggests that the knife was used to cut pasta or potatoes, perhaps.

    The lack of controls is also a serious problem for the prosecution. Even if Dr. Stefanoni did them, she did not report them. Negative controls have been faked (see the article “Tarnish on the Gold Standard,” by William Thompson), and checking the negative control runs for problems is one reason why the release of the electronic data files is so important.

    • maundy says:

      Hi Chris,

      Yes, I can agree that Knox carrying Kercher’s DNA into the flat is a possible route for secondary transfer. Amongst others. In theory. It needs to be kept in mind, though, that the flat was fairly thoroughly searched for DNA traces (about as thoroughly, it looks like, as would be expected in this type of case) and no other transfers from the victim were identified (although, yes, I’ll concede that not every sample seems to have been turned up to 11). No other trace from the victim was found.

      So, the scenario suggested here is that Knox innocently had a tiny bit of innocent DNA from Kercher on her fingers which stubbornly stayed there until they came into contact with – of all the things she could have touched – a knife. And not just that, the very spot on the knife where there was a scratch in the metal, making it just the place where the forensic team would look.

      It’s not impossible, but I think that if courts start accepting that kind of speculative scenario it could be the end of all DNA evidence.

      I agree that the lab contamination is a lot less speculative. As I say above, this mainly comes down to the court getting an account of what the lab procedures were. My hunch is that documentation giving a good picture of this probably does exist. But will it be produced as evidence?

      I don’t think the lack of blood on the knife is a problem in itself. The thing is, if we adopt the hypothesis that the DNA trace was on the knife prior to analysis, what we don’t know is what biological material was present. The physics of how a tiny piece of skin or muscle could become trapped in a scratch on the knife are very easy to appreciate. Anyone who’s had food trapped between their teeth can understand how blood might be easy to remove but a piece of something more substantial could get left behind.

      I agree that controls look like they may turn out to be a problem for the prosecution, but I’m not sure how the faking of controls is an issue here. If Stefanoni is not able to produce any evidence of controls then it would seem pretty clear that she hasn’t taken the trouble to fake any controls.

      • Hi Maundy,

        My point about negative controls is that the defense expects to review them to see that they indeed show no DNA. If the forensic police did controls but never reported them, it is almost as bad as not doing them. If DNA evidence were accepted without negative controls, it would substantially weaken the concept of discovery, IMO.

        We are probably talking about ten cells worth of DNA, so I am not sure that I buy into your food getting stuck into teeth analogy. How would those ten cells avoid getting lysed by osmotic stress or detergents during cleaning?

      • maundy says:

        I think it depends what you mean by “almost as bad”. In terms of analytical procedure, I agree it would not be great. But I also think the difference between running controls and not running controls is pretty big, even if you fail to keep records.

        How would the cells avoid getting lysed? Because sometimes that’s what happens, or else every clean would be a perfect clean. And the likelihood will vary according to what stresses you imagine the material was placed under. Plus, how do we know that there was no cytolysis? The originating tissue (if we make that assumption) could have included a number of burst or damaged cells, it could have been separated from a larger body of tissue at some point. We just can’t account for its history.

        We can also guess as to its size. About ten cells? I’d say it has to be at least that, but we have no real way of guessing at an upper limit. We also can’t be sure how they were arranged. And the important point is we don’t know the dimension of the scratch (at, least, I haven’t been able to find that information). It’s not really possible to say whether a piece of tissue of unknown size can or can’t get lodged in a crevice of unknown size. Given that the material was recovered from a crevice, though, I don’t see why it can’t be a reasonable hypothesis to supposed that it was lodged there.

  5. Hi Maudy,

    One other point about the controls. They would have to be done with the same threshold as the knife profile to be valid.

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