UNDERSTANDING THE INDEPENDENT DNA EXPERTS’ REPORT IN THE AMANDA KNOX CASE (PART I)

  1.   Introduction

On July 30, 2011 the independent DNA experts, Stefano Conti and Carla Vecchiotti will be cross-examined on their famous 145 page report that has given Amanda Knox great hope in her appeal.  The report has been partially translated in a blog at http://knoxdnareport.wordpress.com/ where it is referred to as “The experts’ report discrediting the DNA evidence against Amanda Knox and Raffaelle Sollecito. Translated into English.”  The Italian police scientist, Patrizia Stefanoni, who performed the original DNA testing, is reportedly threatening to sue the experts for false statements regarding her work.  The translated portions of the Conti-Vecchiotti report focus on the knife and the bra clasp.  Originally, the knife was reported to have DNA consistent with Amanda Knox on the handle and a trace amount of DNA consistent with Meredith Kercher on the blade.  No blood was detected on the blade.  A mixture of DNA was found on the bra clasp with the major contributor being consistent with Meredith Kercher, and Raffaele Sollecito not being excluded as a possible minor contributor to the DNA mixture.  The actual source (skin cells, perspiration, blood, saliva, etc.) of the DNA on these items was not determined.

The assignment given to the independent DNA experts by Judge Claudio Pratillo Hellmann is described as follows:

Having examined the record and conducted such technical investigations as shall be necessary, the Expert Panel shall ascertain:

  •  ”whether it is possible, by means of a new technical analysis, to identify the DNA present on items 165b (bra clasp) and 36 (knife), and to determine the reliability of any such identification“
  • “if it is not possible to carry out a new technical analysis, shall evaluate, on the basis of the record, the degree of reliability of the genetic analysis performed by the Scientific Police on the aforementioned items, including with respect to possible contamination.” 

In this blog posting I endeavor to present, summarize, and hopefully shed light on key findings. On occasion, I have injected my two cents worth as well.  Standard disclaimer: I am not a formal expert on the case, I do not have access to all of the data, etc., etc., etc. I hope that this blog posting assists in the understanding of the DNA issues in the Conti-Vecchiotti report.

2.  Evaluation of Knife

The examination of the evidence began on February 9, 2011with a whole host of expert ‘spectators’.  The following consultants were listed as being present while Conti and Vecchiotti performed their analysis:

“-Dr. Patrizia Stefanoni, consultant for the Prosecutor’s Office [Procura Generale];
-Prof. Giuseppe Novelli, consultant for the Prosecutor’s Office;
-Dr. Emiliano Giardina, consultant for the Prosecutor’s Office;
-Prof. Francesca Torricelli, consultant for the civil plaintiff [i.e. the victim’s family];
-Prof. Adriano Tagliabracci, consultant for Raffaele Sollecito;
-Dr. Valerio Onofri, consultant for Raffaele Sollecito;
-Prof. Carlo Torre, consultant for Amanda M. Knox;
-Dr. Sarah Gino, consultant for Amanda M. Knox;
-Dr. Walter Patumi, consultant for Amanda M. Knox.”

The knife (Item 36) was described as having several narrow streaks on the right side that proceeded from the edge of the blade to the halfway point of the blade.  The knife is reported to have an approximately 18 cm long blade and a black plastic handle. Numerous streaks are noted on the blade and a dark-colored material is noted to be present where the blade and handle meet.  Using photographs from the original analysis, areas previously examined were chosen for re-analysis.  Areas “A”, “D”, and “F” were sampled from the handle and areas “B”, “C”, “E”, and “G” were sampled from the blade of the knife.  Two additional (not previously tested) areas “H” and “I” were taken from the point of contact of the blade and handle.

The areas were first tested for the presence of blood using a product called “Combur Test® E” (pictured below).

The test strip is moistened with distilled water and rubbed against the area to be tested.  A quickly appearing blue-green color indicates a positive result.  This is only a presumptive test for blood and is not species-specific.  A positive test result can only indicate the possible presence of blood since false positives can occur.  A negative result is significant since these types of tests while not being very specific, are in fact very sensitive.  These types of tests are used in a clinical setting to detect trace or ‘occult’ blood in places where it should not necessarily be, such as in urine, and are therefore appropriately sensitive.  In a forensic setting, if a positive test result is obtained; further testing is required to confirm the presence of human blood.

All areas tested for blood were negative.  The areas were also sampled for DNA testing using sterile swabs moistened with sterile water.  A small portion of each swab was sampled for microscopic evaluation for cellular material.

The bottom line results for the knife were that insufficient/no amounts of DNA for analysis were obtained for all samples and no identifiable cellular material was observed microscopically.  Granules of starch were observed microscopically on some of the swabs.

3.  Meaning of Results From Knife

When attempting to re-analyze biological evidence for which there is no visible staining, it becomes difficult to make ‘apples and apples’ comparisons between the original and subsequent analyses. For example if a relatively large bloodstain is involved, both parties can test areas where there is known cellular material and similar results would be expected if both tests were carried out correctly.  In the case of the knife, there are only some streaks and dark material that may resemble blood, but were in fact shown to not be blood. Thus, the sampling is almost random as no biological material was identified, and tries to pick up any invisible cellular material that may be present.  Obviously, the first time the knife was swabbed some cellular material may have been removed from the knife.  Based on the low level DNA profile (of unknown biological source) obtained by Stefanoni originally on the blade it can be predicted that if DNA were present on that knife in the area sampled, it was likely completely removed by the first swabbing and there is no way to predict whether similar cellular material might be adjacent to the first swabbing and thus picked up in the second sampling.  This would also potentially explain why the second testing did not replicate the finding of Amanda Knox’s DNA on the handle of the knife-it may have simply been consumed in the first analysis.

It is however significant, that two laboratories tested various areas of the knife (including the new areas at the handle/blade intersection) with sensitive means, and absolutely no trace of blood was detected.

The presence of starch molecules in my opinion is of no significance since there are many sources of starch in our everday environment.  This is particularly not surprising for a knife found in a kitchen.

The absence of any defined cellular material upon microscopic examination in my opinion is not significant. The fact that insufficient DNA for typing was obtained illustrates that there is at best ‘background’ DNA on the knife. In cases where “touch DNA” is being analyzed, such as on the handle of a knife it is impossible to determine the actual cellular origin of the DNA as it could be from skin cells, perspiration, trace amounts of saliva, etc.  Cells from all of these sources cannot be practically differentiated under the microscope and may be few and/or damaged and thus not identifiable.  DNA testing is a more sensitive means to determine if cellular material is present on an object.  Microscopic examination is typically most helpful in identifying sperm cells, which have distinctive morphology.

Since no further DNA testing could be performed, the original testing results and data were later reviewed by Conti and Vecchiotti.

4.   Evaluation of Bra Clasps

The bra clasps were described to have multiple areas of dark-red color (presumably rust) on them.  Two areas “L” and “M” were tested for blood (as described for the knife) with negative results. The areas were then sampled for DNA in the same manner that the knife was sampled.  A negative control consisting of a sterile swab moistened with sterile water was prepared.  The purpose of the negative control is to show that the swabs and water used to take DNA samples with are DNA-free.

No DNA was obtained from areas “L” and “M”. No identifiable cellular material was detected microscopically.

5.  Meaning of Results from Bra Clasps

If the bra clasps were not stored properly to the point that they were allowed to rust, then any remaining DNA was likely destroyed. Also, if the clasps were thoroughly swabbed during the first analysis, any cellular material present may have been removed during that process.  Blood was not detected.

Since no further DNA testing could be performed, the original testing results and data were later reviewed by Conti and Vecchiotti.

(The bigger issues with the bra clasps seem to be how/when they were collected, how they were handled, and how they were improperly preserved.)

See also CBS news report @ http://www.youtube.com/watch?v=l3RN21G72dw

6.  Review of Stefanoni’s Analysis of the Knife

The report starts by quoting transcripts of testimony from Stefanoni in which she describes her logic for sampling of the knife:

“In the G.U.P hearing (04.10.2008) and in the Court of Assizes (hearing on 23.05.2009),  Dr. Stefanoni explained that no traces visible to the naked eye were present on the knife under examination, and that the samples were taken from parts of the [knife] where, based on her own experience, it might have been possible to find biological material. (G.U.P hearing on 04.10.08, pages 17-18: “with regard to sample A, nothing was visible; however, our experience and common sense led us to collect [a sample] there…sample B was not just by chance…the sample was taken from that point because, again only from a visual inspection, that is, with no instrument…streaks were visible to the naked eye”. Court of Assizes (record of the 23.05.09 hearing, page 81): “these scratches were the only element which could, so to speak, guide me in a sample which otherwise, as happened later, was completely random; the other samples which were taken from the knife, therefore I’m referring to…also to C, which in fact is contextual with A and B as samples, but also in particular those which followed, E and G, were done more or less in a random manner because there was no fact, no element which would have led me to take a sample at this point, say, rather than another“.

Court of Assizes (record of the hearing on 23.05.09), page 94): “sample B was collected at this point on the basis that no significant biological trace was visible, so to speak, to the naked eye. [Sample] A was taken at that point, naturally on the handle, as also with D and F, with the intention of possibly finding DNA from the person who had handled that weapon [arma]“.”

Three samples were taken from the handle of the knife, “A”, “D”, and “F” and four samples were taken from the blade “B”, “C”, “E”, and “G”. The samples were reportedly coded and described as follows:

Sample A (“presumed exfoliation cells, letter A”) = 47329

Sample B (“presumed haematic substance, letter B”) = 47330

Sample C (“presumed haematic trace, letter C”) = 47331

Sample D (“presumed exfoliation cells, letter D”) = 48649

Sample E (“presumed haematic substance, letter E”) = 48651

Sample F (“presumed exfoliation cells, letter F”) = 48654

Sample G (“presumed haematic substance, letter G”) = 48655

All of the samples from the blade were tested with a presumptive test for blood as well as a human species-specific test.  All tests were negative indicating that no blood was detected.  The samples from the handle of the knife were not tested for blood. Conti and Vecchiotti point out that the designations of “presumed haematic substance”, are presumptive indeed based on the test results.

No further testing for biological material was conducted, so it is impossible to say what the biological source of any DNA detected on the knife is, other than the fact that no blood is present.  Note that testing for DNA is more sensitive than tests that attempt to identify bodily fluids/cellular material.  Thus, very often laboratories obtain DNA testing results that cannot be positively attributed to a specific biological source.  The great sensitivity of DNA testing can make its interpretation very difficult as trace amounts of DNA can be transferred in many ways.

The table below from Stefanoni’s report indicates that samples A-G were assessed for quantity of DNA using a real-time PCR protocol, however the specific chemistry used is not specified.  Real-time PCR is considered the state of the art means of determining the amount of testable human DNA in forensic samples. The chart indicates that testing for human DNA is positive for samples A and B, and negative for samples C-G.

Conti and Vecchiotti note that samples A-C were actually not tested with the same real-time PCR technique described in the table above. Instead, a ‘Qubit Fluorometer’ with the ‘dsDNA HS Kit’ was used.  According to Conti and Vecchiotti, this method is not specific for human DNA.  I am not familiar with this technique and do not know why it would be used in favor of the standard real-time PCR methodologies.

The results from the Qubit Fluorometer indicate that no DNA was detected in Samples B and C, and that .08 nanograms per microliter of DNA (not necessarily human) was detected in Sample A.

There is therefore a major discrepancy here beyond possibly not reporting the right technique.  Why is Sample B reported as being positive for DNA if none was detected in the assay?  No DNA was detected for C in the same assay, yet C was reported as being negative for DNA in Stefanoni’s report.

Conti and Vecchotti further state:

“Nor is it comprehensible, considering the negative results on sample B, what Dr. Stefanoni reported during the GUP questioning (page 178) where she stated that the DNA in sample B, quantified with Real Time PCR (it is recalled that such quantification confirmed during the hearing was never carried out or, at least, no documentation was provided to support this claim), was in the order of some hundreds of picograms”, a value which does not appear in any of the documents provided to us (SAL, Fluorimeter report, Real Time report, RTIGF).”

To tie all of this in to the final result, sample B is from the knife blade and reportedly has a trace level of DNA consistent with Meredith Kercher on it.  Sample A is from the knife handle and reportedly has DNA consistent with Amanda Knox on it.

Sample A certainly had an amount of DNA amenable for typing and the final result is as expected with regard to level of DNA detected in the final typing.

There is apparent confusion regarding Sample B, which is best explained in the Conti-Vecchiotti report:

“We learn from the transcript of the GUP questioning that Dr. Stefanoni concentrated the volume of the extract from sample B several times.

In particular, she stated first having concentrated the extract from an initial volume of 50 microlitres “to around 20, 22, 23 microlitres” (GUP page 178), and of having subsequently carried out Real Time quantification of the total [amount of] DNA, and not of the DNA of masculine origin.

Since from the Real Time quantification (never carried out!) she obtained a concentration of “some hundreds of picograms of DNA” (GUP, page 178) she took steps to further concentrate the extract in order to obtain a final volume of 10 μl which she would have used for the PCR reaction.

We hold that quantification was also not performed on the final volume, since there is no confirmation either in the documentation in the case file (SAL, Real Time report, RTIGF) nor was such a circumstance ever reported by Dr. Stefanoni in the course of her questioning.

In practical terms, an amplification was carried out without knowledge of one of the basic parameters: that is, the concentration of the DNA possibly extracted from sample B.”

It sounds like Conti-Vecchiotti were not provided with data on this concentration and only learned about it by reviewing transcripts.  Certainly this issue will be vetted in court to determine if Conti-Vecchiotti simply misunderstood the documentation, were not provided with documentation, or if something else is going on. If sample B underwent concentration procedures, it is important that appropriate negative controls were also concentrated to demonstrate that the procedure did not introduce contamination.

At any rate, it appears that Sample B had to be concentrated multiple times, and it is unclear how much, if any DNA was actually introduced into the final typing procedures.

Sample A-handle of knife

The final typing result is below.  Unfortunately, the quality of the scan is poor.  It basically shows a DNA profile of reasonable amplitude based on the quantitation result of .08 nanograms per microliter.  The DNA profile appears to be a single source  DNA profile from a female.

Sample B-Blade of Knife

The final results are displayed below (poor scan quality).  There were two sets of results for the sample.



It is immediately obvious to anyone familiar with these electropherograms, that this is a very low level result.  The taller the DNA peaks present, the more DNA present.  Many of the peaks are below a height of 50 RFU (relative fluorescence units) and do not have a classic triangular DNA peak shape.

I have actually never seen a laboratory in the U.S. report peaks below 50 RFU.  In fact, I believe that the manufacturer of the DNA typing chemistry and software used in this case actually recommends setting the threshold at 50 RFU for basic peak detection.  The problem when you consider peaks that low is that they can be confused with instrumental noise.  You can see that smaller peaks have a more jagged appearance.

If in fact Stefanoni’s laboratory performed thorough validation studies that demonstrated the parameters under which such low peaks could be reported, then there may be some justification for this practice. Certainly, great caution would need to be used with that sort of interpretation, and its meaning is open to great debate.

This less than robust result coupled with the confusion regarding the DNA quantitation steps raises many questions about the quality of the result. When contamination occurs in DNA laboratories, it is often at a very low level. It will be very interesting to see what Stefanoni’s explanation for reporting such a low result is.

In summary, there are a few possible scenarios to consider:

-Based on the confusion with regards to the quantitation, potential questions emerge about the authenticity of the analysis.

-Is the DNA profile even reportable according to the laboratory’s guidelines, and….are such guidelines supported by appropriate validation studies?

-The DNA is actually a contaminant and was not present on the knife.

-The DNA was actually present on the knife.  It clearly cannot be associated with blood.  At the level that the DNA was detected, there are numerous possibilities for how the DNA may have gotten on the knife via innocent transfer.

It is certainly safe to say that the result is in no way consistent with blood from Meredith Kercher being present on the knife.

7.  Review of Stefanoni’s Analysis of Bra Clasps

(To be addressed in Part II of this posting at a later time)

Further Reading:

http://knoxdnareport.wordpress.com/

http://www.roche.com/products/product-details.htm?type=product&id=39

http://www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qubit.html

https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601705

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12 thoughts on “UNDERSTANDING THE INDEPENDENT DNA EXPERTS’ REPORT IN THE AMANDA KNOX CASE (PART I)

    1. Chris,

      Thank you for checking out my posting.

      It must be remembered that concentrating DNA extracts introduces additional opportunities for contamination. The more that tubes are manipulated, the more instances in which they could become contaminated. Also, when DNA is concentrated, any possible trace contaminant is also concentrated and can become more prevalent in the sample.

      For these reasons, the negative extraction control is typically (and in my opinion this is mandatory) subjected to the same concentration steps to demonstrate that there is not low level contamination in the reagents and that the additional manipulations did not introduce contamination. If the negative control is clean after being concentrated, then greater confidence can be attributed to the results. I wonder if this step was performed in this case.

  1. Excellent article. Thank you.

    I have read that during the cross examination Vecchiotti was forced to admit that it is *possible* the DNA profile is Kercher’s. I wish she had added that it could not possibly be a profile of Kercher’s blood. (That’s clear if one knows the details, but still would have been a good point to emphasize).

    I, too, look forward to part 2.

    1. Thank you for reading it. I wanted to make one note on the identification of human blood. In certain instances, typically due to limited quantity of a suspected blood stain, confirmatory testing is not possible. Short of performing a confirmatory test, the factors to consider when deciding how likely it is that the stain is human blood are: a) does it have the appearance of blood? b) did it give a positive presumptive test for blood? c) did it yield an amount of DNA that you would expect for a bloodstain of that size? d) if unstained areas were sampled adjacent, did they yield significantly less than the suspected stain area?

  2. You make a point that I have not seen elsewhere.

    Sample B is the only sample of interest. If I understood correctly, the documentation C&V received from Stefanoni showed sample B was NEGATIVE for DNA.

    I realize concentration procedures and positive/negative controls are important — but only if the DNA exists, right? If there was no DNA on the blade, there’s no DNA to discuss unless Stefanoni can prove sample B had DNA.

    Yet people continue to discuss sample B, so I must not have understood correctly. Obviously, there’s a difference between: A) Not providing documentation; and B) Providing documentation that shows sample B tested negative for DNA. Seems to me if the latter occurred, other issues are moot.

    Would you mind explaining how lab documentation is typically produced? I would think DNA test equipment generates electronic files with time stamps — and log files that show the number and order of tests. But perhaps I’m wrong, and it is standard practice to record results (or not) by hand.

    I did understand there was no presumptive indication of blood; hence nothing to subject to confirmatory testing. To me, that alone rules out the knife as murder weapon.

    1. According to C&V, the only documentation they had was that when the original DNA extract for B was submitted to quantitation procedures it gave a negative result for DNA according to the notes, although the report said that B was positive for DNA. They noted that in transcripts Stefanoni mentioned that she carried out concentration procedures on the original extract and performed real-time PCR quantitation that revealed a low level of human DNA in the concentrated extract of Item B. If that is what truly occurred the only thing I can think of that explains the discrepancy between the notes C&V reviewed and the report is that they did not have the notes on the concentration or real-time PCR quantitation. If these notes do not exist, then all bets are off-everything must be documented for the analysis to have validity. This is a big issue and I would assume that C&V would have pressed to see if these notes on the concentration and real time PCR exist. If they didn’t press on this issue, then they may be jumping to conclusions.

      It can be difficult to obtain full discovery on DNA testing. However, if there are specific requests for information that are not turned over without explanation, then as a DNA consultant I can only assume that the records requested do not exist.

      Another issue to consider is that the final DNA typing (PCR and capillary electrophoresis) may sometimes reveal trace amounts of DNA that were not detected in the quantitation step. This just has to do with the greater sensitivity of the final DNA typing process.

      Typical DNA discovery in the U.S. includes the analyst’s hand written notes, sketches, photographs, instrument printouts, and raw electronic data from capillary electrophoresis. For instrumental procedures, there are absolutely printouts with dates, times, sample names, etc. There are often handwritten notes complementing and summarizing instrumental printouts.

      I typically ask for the entire casefile including all reports, notes, data, photographs, communication logs, chain of custody information, and evidence collection, screening, and preservation notes. Also, the CV of all personnel involved, proficiency test records, any documentation regarding QC issues with the case, all electronic STR data, protocols, quality manual, and interpretation guidelines.

  3. Conti and Vecchiotti, the independent experts appointed in the trial of second instance (something akin to an appeal trial) resampled the knife (the highly disputed murder weapon) and found small amounts of DNA when they ran real-time quantification. Sample I provided the most DNA, and it was 5 pg/µL. To the best of my knowledge every test for biological substance (including blood) came up negative in both this work and the original work.

    If one looks at the standards they ran (starting around page 20 in the translated report) in the real time experiment, they range from 50 ng per µL to 0.023 ng per µL ( which is 23 pg per liter). The concentration from sample I was 0.005 ng per µL. This is between 20 and 25% of the value of the lowest point on the standard curve. I would question how well we even know the 0.005 ng/µL number, because it is an extrapolation below the lowest point from the standard curve. My question is whether Conti and Vecchiotti were correct to terminate the experiment at this stage. My understanding is that they did so with the assent of everyone who was present.

    1. Hi Chris,

      Great questions. No doubt that values in that range are inexact. It sounds like it was not unreasonable to not pursue amplification based on that quant result. Depending on the lab/instrument/chemistry known blanks may give some sort of ‘noise’ result in the range of .005 ng/ul. Labs will often conduct studies to figure out what a true ‘no amplifiable DNA’ quant result is. It may be that based on the studies of the laboratory, there was no reason to believe that .005 ng/ul would give any type of useful amplification product. The other thing to consider is the volume of the extract. For example if a 20ul extract gave this result, there isn’t much room to concentrate for amplification. If however, the extract were 100ul, then concentrating down to 10 ul may significantly increase the chances of detecting something after PCR and capillary electrophoresis.

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