Going to give this a shot wasnt_me. What I will try and do is give a brief overview then give backup info from the experts report all from the same link which is provided in your post. (My comments are bolded)
We know at this point that the knife tested negative for blood and Stephanoni received a too low reading for both sample b and c and is below the 50 rfu threshold but she decides that sample b had biological material. As you can see from their report the experts are perplexed
"On the other hand, it is not possible to comprehend the criteria adopted in the assessment of the positive quantification result for sample B and the negative result for sample C, given that the same result, “too low”, was obtained for both samples: that is, a value which must be considered not only below the sensitivity threshold of the Fluorimeter indicated by the manual (DNA concentrations equal to 0.2 ng/μl), but below 0.08 ng/μl, a value which the Fluorimeter detected for sample A."
From the SAL's they know that the extraction was on a specific date as is stated below:
"Extraction of DNA
"The Work Status Report (SAL) shows that:
• the extraction of DNA from samples A-B-C was performed on 13.11.07;"
We then find out that she did not use Real Time quantification but Qubit Fluormeter without knowing what the concentration of the DNA is in sample b. so the theory presented by Dr. Gino appears to have been taken up by Stephanoni when Dr. Gino testified that it was LCN DNA and that she only performed the amplification once which it appears she knew was not acceptable
" Amplification
• On the other hand, considerable perplexity arises about the quantity of DNA extracted from sample B which would have been added to the reaction mix. The conditional is necessary [1], in that the extract from sample B was quantified using the Qubit Fluorometer™ and although having given an uninterpretable result (“too low”
was considered “positive”, in contrast to sample C which, although being “too low”, was considered negative!
• 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 having first 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 “a few hundred 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 consider that quantification was not even 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 performed without knowledge of one of the basic parameters: that is, the concentration of the DNA possibly extracted from sample B.
• What is surprising is that, given the delicate nature of the testing, quantification of samples B and C was not carried out with Real Time – as happened for samples D-E-F-G – to ascertain if a Low Copy Number (LCN) sample was being dealt with, for which a different and more complex procedure should have been adopted.
• The theory that this was a sample which could have been considered LCN was proposed by Dr. Gino in the course of the GUP hearing, and was endorsed by Dr. Stefanoni (“Yes it is possible, certainly!”, page 178) who, although conscious of the problems associated with samples of low DNA quantity (Low Copy Number), did not consider it appropriate to apply any of the precautions recommended by the scientific community in the case of LCN.
• It is emphasized that the amplification was performed only once (pages 21-22, transcript of the GUP questioning. To the question, “…the testing of a trace of this sort should be repeated several times to be considered reliable?” The Technical Consultant replies: “In theory yes”. To the question, “How many times did you do it?” she responds, “In this case only once”."
We then know that the experts required more information which Stephanoni did not want to turn over until ordered to do so. The experts note that the peaks are well below 50 rfu. The experts then find out that she did not repeat the amplification and instead did 2 runs of the same amplification in which they note allele loss/additional peaks. Stephanoni did not perform either the negative control or the positive control which is critical. The experts then feel that it is important to present some information with respect to issues related to LCN DNA
Capillary Electrophoresis
"Since the electrophoretic graphs, produced both in CD-ROM and e-mail format, do not differ from each other in any significant way (c.f. peak heights), for ease of exposition our comments here relate to the electrophoretic runs dated Sept. 23 2008, 10:35 AM and Sept. 25 2008, 01:17 PM, but are equally intended to apply to the electropherograms dated May 11 2011, 04:48 PM and May 11 2011, 04:36 PM.
From an examination of Electrophoretic Run 1, dated “Sept. 23 2008, 10:35 AM, it must be noted that: 1) this graph shows peaks which are clearly below the 50 RFU threshold (it is emphasized that 50 RFU is the threshold recommended by the kit manual, and it is not advised to go below this point); b) the alleles are imbalanced in that the ratio between many peaks is <0.60 (Gill P. et al., 2006).
Regarding the assessment of the peak heights in sample B, the Technical Consultant was specifically asked (GUP, page 20) “When in your experience do you define an RFU value as rather low?” and answered: “below 50, I start to take greater care in the assessment”. Responding to the explanations requested by the GUP (page 21) about the sample with identification code 47330 (sample B, Exhibit 36) – “Perfect! What do we have here?” – the Technical Consultant replies: “Well, here we have a genetic profile which certainly has a lower R.S.U. [RFU?] intensity than the previous one”. Q: “In the order of…?” A: “Let’s say we’re in the order of, let me see, again the same genetic point that I defined earlier, at which we see a 41 and a 28”. Q: Therefore for you there would already be a bit of a risk, as we said before a little bit low?” A: “Yes, yes, yes”.
Again, responding to a question from Dr. Biondo (transcript of the GUP hearing, page 72): “There was another aspect for which, when one does a DNA analysis where the peaks, the heights reported in R.S.U. are low, you said there was a risk. Can you explain this risk?” The Technical Consultant replies: “Well, there’s a risk in the sense that in practice when I evaluate a genetic profile which has rather low peak heights, roughly below 50 RFU, 50, 60, I risk performing an operation which is something, so to speak, that no forensic geneticist would wish to do, and that is to poorly interpret that profile…that is, in the sense that I might make a mistake in the assessment, assess a peak in an erroneous manner because perhaps it is too low, and therefore perhaps run the risk of excluding a person who might be the victim from the framework of the investigations…”
The following question is reported on pages 21-22 of the GUP questioning: “…the testing of a trace of this sort should be repeated several times to be considered reliable?” The Technical Consultant replies: “In theory yes”. To the question, “How many times did you do it?” she responds: “In this case only once”. Q: “Just once, and therefore in theory why ought it to be considered more reliable if it is done several times?” A: “Because reproducibility of the result is, let’s say, a good standard in any scientific experiment quite apart from forensic genetics, obviously to be considered valid a result must be repeatable”.
In fact, the Technical Consultant did not repeat the amplification of the extract but performed two electrophoretic runs of the same amplification. From a comparison of the two separate runs, the existence of peak imbalance and inversion is immediately obvious, to the point where in some cases there is allele loss or the presence of an additional peak (c.f. electrophoretic graphs, runs 1-2, Sept. 2008).
In addition, it must be noted that neither the negative control – which, as previously mentioned, could have indicated the presence of possible contamination – nor the positive control, which would have allowed the effectiveness of the selected experimental conditions to be monitored, are present in the electropherograms produced.
From what has previously been stated about the analysis of the electropherograms relating to sample B, it is inferred that the quantity of extracted and amplified DNA must have been very low, falling within the definition of Low Copy Number (LCN) or Low-Template DNA (LT-DNA).
It is considered appropriate at this point to provide some clarifications about the definition of the term Low Copy Number, and to outline the experiences, suggestions and recommendations of the International Scientific Community aimed at trying to resolve some of the most frequent problems associated with biological samples of low DNA quantity (LCN)
Issues noted with respect to LCN DNA by the experts are as follows
"Low Copy Number (LCN) or Low Template DNA (LT-DNA)
• greater potential risk of error in comparison with conventional STR typing protocols;
• errors of interpretation due to allele drop-in, allele drop-out, peak height imbalance and large stutter peaks;
• the need for a robust and reliable quantification method in order to determine the amount of DNA available for analysis;
• LCN profiles are not generally reproducible and, due to the potential for error, the probative value of the results may not be evaluated correctly;
• the interpretation of profiles derived from mixtures using LCN typing is problematic, and at the moment there are no interpretation guidelines based on reliable validation studies;
• due to the sensitivity of the method and the types of samples analyzed (for example, “touch” samples), the LCN profile may not be relevant to the specific case;
• the evidence cannot be used for exculpatory purposes;
• instructions about the proper collection of items and protocols regarding their handling have not been clearly established or communicated;
• reagents, consumables and laboratory instrumentation can contain low levels of extraneous DNA which may complicate the interpretation of LCN typing results"
In the conclusion the experts note that DNA was not revealed on the knife but they did find starch. It as well tested negative for bleach.
Conclusions (2)
"- The cytomorphological tests on the items did not reveal the presence of cellular material. Some samples of item 36 (knife), in particular sample “H”, present granules with a circular/hexagonal characteristic morphology with a cental radial structure. A more detailed microscopic study, together with the consultation of data in the literature, allowed us to ascertain that the structures in question are attributable to granules of starch, thus matter of a vegetable nature.
- The quantification of the extracts obtained from the samples obtained from item 36 (knife) and item 165B (bra clasps), conducted via Real Time PCR, did not reveal the presence of DNA.
- In view of the absence of DNA in the extracts that we obtained, with the agreement of the consultants for the parties, we did not proceed to the subsequent amplification step
ITEM 36 (KNIFE)
Relative to the genetic analysis performed on trace A (handle of the knife), we agree with the conclusion reached by the Technical Consultant regarding the attribution of the genetic profile obtained from these samples to Amanda Marie Knox.
Relative to trace B (blade of the knife) we find that the technical analyses performed are not reliable for the following reasons:
1. There does not exist evidence which scientifically confirms that trace B (blade of knife) is the product of blood.
2. The electrophoretic profiles exhibited reveal that the sample indicated by the letter B (blade of knife) was a Low Copy Number (LCN) sample, and, as such, all of the precautions indicated by the international scientific community should have been applied.
3. Taking into account that none of the recommendations of the international scientific community relative to the treatment of Low Copy Number (LCN) samples were followed, we do not accept the conclusions regarding the certain attribution of the profile found on trace B (blade of knife) to the victim Meredith Susanna Cara Kercher, since the genetic profile, as obtained, appears unreliable insofar as it is not supported by scientifically validated analysis;
4. International protocols of inspection, collection, and sampling were not followed;
5. It cannot be ruled out that the result obtained from sample B (blade of knife) derives from contamination in some phase of the collection and/or handling and/or analyses performed."
http://knoxdnareport.wordpress.com/contents/conclusions-2/
