From what has been set forth above, we hold that the documentation in the case file reveals inaccuracies and gaps about the analytical procedures performed on Exhibit 36.
– regarding the nature of the material collected, there is no scientifically conclusive evidence to support the possible blood nature of sample B (knife blade) in that both the generic blood test and the human species test were negative.
The existence of presumed exfoliated cells on the samples taken from the knife handle is equally lacking in scientific basis.
Therefore we repeat that the theories formulated by the Technical Consultant about the nature of the material removed from Exhibit 36 are wholly arbitrary in that they are not supported by any objective confirmation.
– It should be stressed that the documentation in the case file relating to the traceability of the analytical operations performed is completely inadequate.
a) regarding the DNA quantification tests, both in the Technical Report on the Forensic Genetic Tests (RTIGF) and in the GUP, the Technical Consultant repeatedly claimed to have performed quantification with Real Time PCR on all the samples taken from the knife, but this claim is contradicted by the documentation produced: in fact, the Qubit Fluorimeter™ was used for samples A-B-C.
Regarding sample A (knife handle) the results obtained (Qubit Fluorimeter™) reveal that the DNA concentration in this sample was 0.08 ng/μl.
Taking into account that the “extract quantity” was 50 μl (c.f. SAL), multiplying 0.08 ng/μl x 50 μl, the total DNA [obtained] was 4ng: certainly a significant quantity, which allowed sample A to be considered positive to quantification.
The amount of DNA used for the subsequent amplification (0.8 ng) falls within the range suggested by the kit (0.5-1.25 ng/μl of template DNA) and provided an electrophoretic graph of good quality, in accordance with the amount of DNA used for the reaction.
On the other hand, it is not possible to comprehend the criteria adopted in the assessment of the positive quantification result of sample B and the negative result of 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 of 0.2 ng/μl) but below 0.08 ng/μl, a value which the Fluorimeter detected for sample A.
Neither is it comprehensible, considering the negative results on sample B, what Dr. Stefanoni reported in 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 as 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).
b) regarding the extract obtained from sample B (knife blade), the Work Status Report (SAL) shows that this extract was 50 μl. In the GUP hearing, the Technical Consultant affirmed having concentrated sample B to “20, 22, 23 microliters”, of having quantified it with Real Time (GUP, page 178: “Quantification which you did with real time, I imagine?” A: “Eh, yes”) and of subsequently concentrating it again to “10 microliters”, but there is no trace of these operations in the documentation exhibited (c.f. SAL). Neither is it known what the amount of DNA was in the extract concentrated to “20, 22, 23 microliters” and/or the amount of DNA in the extract concentrated to 10 μl.
The quantification problem is of fundamental importance, since a DNA quantity of less than 200 pg/μl falls within the Low Copy Number (LCN) definition, for which the International Scientific Community recommends protocols aimed at obtaining scientifically reliable results.
Since the problems arising from the testing of sub-optimal quantities of DNA are numerous (peak imbalance, drop-in, drop-out) different authors have proposed largely interchangeable scientific approaches which can make the interpretation of the data obtained easier.
It should be emphasized that what all the protocols proposed have in common is an awareness that the main problem of LCN samples is contamination of the item: therefore the authors unanimously agree that suitable protocols in the inspection procedures must be applied in order to minimize environmental contamination at the crime scene, and strict protocols for the collection and sampling of items in order to minimize contamination from handling at the crime scene.
The procedures recommended by all to reduce laboratory contamination are equally rigorous (decontamination of the environment, suitable protection of the operatives, control of the reagents employed in procedures, etc.) as it is well-known that contaminant DNA at low levels may derive from reagents and other laboratory consumables, from the staff, and from cross-contamination from sample to sample.
It should also be noted that, according to the International Scientific Community, even if the protocols of a general character indicated above are scrupulously applied, the increased sensitivity of the PCR method (obtained by modifying the standard amplification protocols, increasing the [no. of] PCR cycles, doubling the annealing time, increasing injection time) nonetheless constitutes a higher potential for contamination; therefore, secondary transfer cannot be excluded as a possible explanation for the results obtained from LCN typing (Budowle et al., 2009).
In addition, the possibility is noted that the PCR product of LCN may show results which are due to amplified DNA contaminating an unamplified sample: this is because, as previously related, amplified DNA is much more concentrated than un-amplified template DNA, and so the first will be preferentially copied during the PCR.
For this reason, the samples being tested should usually be worked on in the laboratory before the reference samples, in order to avoid any possibility of evidence contamination with already amplified DNA.
In the case in question, it is recalled that Exhibit 36 was placed for testing into a context where a considerable number of samples belonging to the victim had already been examined; therefore, it cannot be excluded that contamination by the aforementioned methods may have occurred – all the more so because the negative controls, which should have been amplified contextually and which could have given an indication as to the absence of contamination, were not produced (Record of the Court of Assizes hearing on 23/05/09, pages 29-30: “Well, the knife was tested as one item in the course of 50 samples attributed to the victim, some were before the tests on the knife naturally, and others after, so of these 50…I don’t know the knife was placed, now I don’t know, at a fourth, a third of this series of tests, but in any case even if the knife was analyzed at the end of these 50, 60 samples, in any case this wouldn’t affect the validity of the results, because each sample is tested separately, it is absolutely impossible to mix one sample with another, also because the Kercher file is just one of many files we dealt with simultaneously in the laboratory, it’s not as if the Scientific Police Service stopped to deal with the Kercher file…”).
Another key aspect concerns the procedure to follow for a reliable interpretation of the results, and for the designation of an allele in an LCN sample. The procedure recognized as valid by the International Scientific Community comes from replicate analysis: that is, it requires the division of the sample into two or more aliquots, with only those peaks recurring in at least two replicates being considered alleles.
The advantage of this approach is that if contamination occurs in a random and infrequent way, observing an allele several times increases the likelihood that it actually derives from the sample being examined, assuming that contamination did not happen during the sampling phase.
Most scientists who work with LCN stress the need to perform 2-3 replications, and state that an allele must be observed at least twice to be denominated as such: to date, allele redundancy is the recognized and accepted methodology, and is the cornerstone of reliable LCN typing: (GUP hearing on 05.10.08, pages 21-22, to the question “…the testing of a trace of this type should be repeated several times to be considered reliable?” the Technical Consultant responds: “In theory yes”. To the question: “How many times did you do it?” she responds: “In this case only once”. Q: “Only once, and therefore in theory why ought it be considered more reliable if one does it several times?” A: Because reproducibility of the result is, so to speak, a good standard in any scientific experiment quite apart from forensic genetics, obviously in order to be considered valid a result must be repeatable”).
Therefore, taking into account that in this specific case:
– it does not appear that inspection procedures were carried out according to international protocols in order to minimize environmental contamination;
– international protocols of collection and sampling of the item were not applied in order to minimize contamination from handling;
– it is not known whether rigorous decontamination procedures were applied in the laboratory to minimize laboratory contamination;
– a reliable method for quantifying the DNA from samples A-B-C was not employed, and the quantification performed with the Qubit Fluorimeter™ gave the result “too low” for samples B-C, indicating a DNA amount below the sensitivity threshold of the Fluorimeter (>200 pg/μl), and therefore indicative of a probable LCN sample;
– the electrophoretic graphs exhibited show that the sample indicated with the letter B (knife blade) should have been considered an LCN sample (peak imbalance, RFU below 50 for most of the alleles) and as an LCN sample, all the precautions indicated by the International Scientific Community should have been applied. Amongst these we recall:
a) rigorous respect for decontamination procedures for the instrumentation, the laboratory and the staff (as already mentioned, the procedures adopted to minimize contamination are not reported);
b) testing of the item in a laboratory where no items ascribable to the victim were tested, to avoid any possibility of evidence contamination with already amplified DNA. On the contrary, it was reported that the item was placed for testing into a context where a considerable number of samples belonging to the victim had already been examined (Record of the Court of Assizes hearing on 23.05.09, pages 29-30: “the knife was tested as one item in the course of 50 samples attributed to the victim, some were before the tests on the knife naturally, and others after, so of these 50…I don’t know the knife was placed, now I don’t know, at a fourth, a third of this series of tests…”);
c) performing 2-3 replicate amplifications with the development of a consensus profile. In the case in question, the amplification was only performed once; therefore the lack of replicate amplification with the development of a consensus profile provides unreliable results (GUP hearing on 05.10.08, pages 21-22: to the question, “…the testing of a trace of this type should be repeated several times to be considered reliable?” The TC responds: “In theory yes”. To the question: “How many times did you do it?” she responds: “In this case only once”. Q: “Only once, and therefore in theory why ought it be considered more reliable if one does it several times?” A: Because reproducibility of the result is, so to speak, a good standard in any scientific experiment quite apart from forensic genetics, obviously in order to be considered valid a result must be repeatable”).
d) employment of negative controls in the amplification procedure to check for the presence of contamination. In the attached eletropherograms, neither negative nor positive controls are reported.
Thus premised, in this specific case the following conclusions can be drawn:
– in relation to sample A (knife handle: identification code 47329), taking into account the considerations previously stated about the electrophoretic graph which shows peaks which exceed the 50 RFU threshold and allele balance (Hb=φa/φb >0.60) in accordance with the presumed quantity of DNA used for the reaction (0.8 ng), we agree with the conclusion reached by the Technical Consultant about the attribution of the genetic profile obtained from this sample to Amanda Marie Knox;
– in relation to sample B (knife blade: identification code 47330), based on the considerations previously stated about the electrophoretic graph which shows peaks below the 50 RFU threshold and allele imbalance (Hb=φa/φb >0.60) indicative of a Low Copy Number sample (LCN), taking into account that in this specific case none of the recommendations from the International Scientific Community relating to the treatment of Low Copy Number samples were followed, we do not accept the conclusions about the certain attribution of the profile detected in Sample B to the victim Meredith Susanna Cara Kercher since the genetic profile, as obtained, is unreliable in that it is not supported by scientifically valid analytical procedures.
Neither, as previously explained, can it be excluded that the result obtained from this sample may derive from contamination phenomena occurring at any stage of the collection and/or handling and/or analytical procedures performed.