Capillary Electrophoresis

The amplification product of the Y chromosome obtained from Exhibit 165B was subjected to capillary electrophoresis by means of the ABIPRISM 3130 Genetic Analyzer using the “Gene Mapper” analysis software.

Since the record does not show that any modification was made to the electrophoresis protocol, we assume that the running conditions were the standard ones indicated by the kit.

We now report the graph relating to the electrophoretic run of the DNA amplified from sample 165B dated “Jun 10,2008 01:06 PM” appended to the RTIGF:

[graph p. 129]

The Technical Consultant concludes that “The analysis of the Y chromosome has permitted the determination of the Y haplotype shown in Table 165-II relative to the DNA extracted from trace B. This result also confirms the presence of DNA belonging to Raffaele SOLLECITO in the analyzed trace, since the Y haplotype obtained is equal to that belonging to Raffaele SOLLECITO (comparison made with the Y haplotype already reported in Table 30-II of p.63, extrapolated from the genetic analysis of the salivary swab taken from the same [person].”

We report below the table summarizing the results obtained by the Technical Consultant for the Y chromosome:

[table p. 130]

Subsequently, Dr. Stefanoni provided us with the electrophoretic graph (May 11, 2011 04:08 PM) of the same run but with the indications relative to the height and the areas of all the peaks present in the aforementioned graph.

In light of the numerical values relative to the hight of the peaks present in the electropherogram, we can make the following observations regarding the interpretation of the alleles performed by the Technical Consultant.

Observation of the electropherogram shows that, besides the peaks indicated in the RTIGF as alleles, additional peaks exceeding the threshold of 50 RFU are present, which despite not being in stutter position were not taken into consideration by the Technical Consultant.

Below, we report the electropherograms with an arrow marking the peaks not reported in the RTIGF:

[graph p. 132]
[graph p. 133]

What has been illustrated above shows that more alleles are present in the electropherogram relative to the Y chromosome than are reported in the RTIGF.

In the following table, the alleles revealed by a careful reading of the electropherogram are summarized:

[table p. 134]

It follows from this that several minor contributors of male sex are present in the DNA extracted from Exhibit 165B, confirming what was already observed in the electropherogram of the autosomic STRs and which was not revealed by the Technical Consultant.

Thus we agree with Dr. Stefanoni’s assertion regarding “the extrapolation of a genetic profile deriving from a mixture of biological substances belonging to at least two individuals, at least one of male sex” but we cannot accept the conclusion stating that “the genetic profile is compatible with the hypothesis of a mixture of biological substances (presumably flaking cells) belonging” only “to Raffaele Sollecito and Meredith Susanna Cara Kercher“.

We find that the Technical Consultant arrived at this conclusion restricted to two individuals (Meredith Kercher and Raffaele Sollecito) following an incorrect interpretation of the electropherograms of the autosomic STRs, as a result of disregarding the recommendations of the ISFG concerning the correct interpretation of mixtures (“Recommendation 6: If the crime profile is a major/minor mixture, where minor alleles are the same size (height or area) as stutter of major alleles, then the stutters and minor alleles are indistinguishable. Under these circumstances, alleles in stutter position that do not support H_p [the prosecution’s hypothesis] should be included in the assessment”) and point 7 of the aforementioned ISFG recommendations (Point no. 7: Drop-out: “The consideration on drop-out is analogous to that on stutter“). Had these recommendations been followed, they would have allowed one to reach the conclusion that several minor contributors were present in the trace besides the victim (major contributor).

The latter assertion is supported by the electropherogram relative to the Y chromosome, where several alleles are present that, despite being particularly evident, were not taken into consideration by the Technical Consultant.

The genetic profile thus derives from a mixture of unidentified biological substances (it will be recalled that no test was performed with a view toward revealing the presence of flaking cells, and so the claim is without scientific basis), whose larger component is represented by the DNA of the victim and whose smaller component is represented by DNA from several individuals (cf. autosomic STRs) of male sex (cf. Y chromosome), of which one of the Y haplotypes corresponds to the Y haplotype of Raffaele Sollecito

Regarding the reliability of the item with specific reference to “possible contamination“, we find it appropriate to examine the means by which and circumstances under which Exhibit 165B was acquired.

The item was recovered 46 days after the crime, in a context highly suggestive of environmental contamination.

The DNA obtained, though sufficient in quantity to permit analysis, does not satisfy the minimum quality requirements, due to the evidence of environmental contamination.

Various peaks (cf. table of autosomic STRs and Y-chromosome haplotype) which should have been considered alleles until proven otherwise, were not taken into consideration in the analyses; yet their presence was indicative of the fact that, besides Kercher and Sollecito, other unidentified individuals were represented in the genetic traces found at the crime scene. In this regard, it was necessary to proceed to further amplifications of the extracted DNA in order to confirm the presence of the various haplotypes present at the crime scene — something which was not done, even though a sufficient amount of extracted material was available (cf. SAL: 50 μL of extracted material).

Furthermore, the documentation regarding possible contamination of the item, both before and after recovery, is inadequate. The mere fact that the amplification control — which was not provided — was negative is not enough to rule out environmental contamination of the item previous to the extraction and amplification of the DNA. It would have been necessary to obtain the allele profiles present in the surrounding environment.

The item was recovered on the floor, where it predictably had contact with ambient dust, which, in closed environments frequented by humans, is composed to a large extent of elements of human origin (cells, hairs, etc.).

It has been demonstrated that dust from closed environments can contain tens of micrograms of DNA per gram, with the amount of DNA depending on the level of frequentation by individuals and on the amount of dust that accumulates in the relevant environment.

It has been thoroughly demonstrated that the presence of ambient dust constitutes a significant source of contamination in forensic investigations, since the DNA deriving from such dust can present itself in the form of alleles in analyses of polymorphisms.

The risk of incorrectly interpreting such environmental contaminants can be minimized only by taking the care [accortezza] to institute extremely strict control protocols  including the analysis of extracts from sterile cotton swabs soaked with sterile buffer that have passed on ambient surfaces to take dust samples (Toothman MH et al., 2008).

In any case, the allele profiles obtained from ambient dust, or from samples contaminated with ambient dust, can be indicative of the individuals who have frequented that environment.

Nevertheless, the aforementioned authors explain that it is difficult to generalize to a direct correlation between human frequentation and levels of dust with the quantity and quality of human DNA therein present, because of the potential effects of other uncontrolled factors. Indeed, environmental variables, including light, heat, and humidity can degrade DNA, and residues of detergents (such as bleach) can destroy the DNA. Moreover, the ventilation system can act as a vehicle for transferring dust among different rooms, introducing DNA from individuals that have not necessarily frequented a specific environment (Toothman MH. et al., 2008).

In order to advance interpretive hypotheses, it would have been necessary, in our opinion, to proceed to multiple amplifications on Exhibit 165B, whose alleles should have been compared with the alleles obtained from multiple amplifications performed on extracts from multiple samples of ambient dust.

Only alleles found on Exhibit 165B, and not in ambient dust, could be considered of possible evidentiary value — independently of the height and area of the corresponding peaks. This [comparison] not having been done, the allele profiles of Exhibit 165B should not, in our opinion, be considered probative.

Moreover, given what has been explained above relative to the inspection methods, having seen the documentation in the record, and in particular the DVD of the filmed investigation of the scene [indagini di sopraluogo], the official photos of the Scientific Police, and the the statements made in court, we find that the universally noted inspection procedures and correct protocols of collection and sampling of items were not performed on Via della Pergola, even [those designed] to prevent [scongiurare] the risk of environmental contamination.

And in particular, conspicuous lackings [palesi carenze] were revealed on the following points:

– Specific areas for the containment of contamination were not delimited, or rather this was done only at the outermost level, without worrying about more internal levels of security including the “crime scene“; this however carries with it the possibility of contamination arising from possible transfers between internal environments of the residence, including the room in which the body of Meredith Kercher was found (crime scene).

– Related to the above, no security corridor was created for internal access with anticontamination criteria between the various environments; and indeed on these first two points, Dr. Stefanoni herself has stated, in the deposition of 10-4-2008 on pp.38-39: “…since we had our foot-covers [calzari], and had been entering many times obviously for the inspection activities in that room, and since the blood was abundant…the floor was certainly dirtier the second time, quote unquote, that is the blood had actually been spread around compared to the beginning of the inspection… this is the point, that is contamination is to be understood as something foreign [esogeno] brought in from the outside onto the crime scene… the only thing that could have happened in general in any inspection is that something could have been transferred which however is already there, if this something was moved, this can’t be ruled out but certainly nothing was brought in from the outside….Q:..So the most you can assume is a movement of DNA already present at the site?…A: Yes…” And again on pp. 83-84: “…Q: However the foot-covers aren’t being changed while you move around the house? A: It was possible to go back in…Q:…you went in and out of the rooms without changing the foot-covers…? A: Yes…”

– There was no appropriate location set up for the storage of disposable material used during the inspections (the wastebasket present in Sollecito’s house was even used); in this sense we emphasize the possible increase in the risk of contamination due to the stock [giacenza] of material used and thus contaminated.

– There were deficiencies in the records of access to the area of the crime, that is a record was not correctly kept of who entered the environment. Not only that; in the immediacy of the event, there was access by personnel of the 118 without any protective clothing, personnel who among other things touched and removed even the duvet which covered the body of Meredith (deposition of Monica Napoleoni, hearing of 2/27/2009, p. 228-229): “… A. I stepped into the room while the lady [dottoressa] from the 118 uncovered the corpse. PM:…when you went in, did you have your suit on? A: …no, I wore foot-covers and sterile gloves… PM: so everyone who went in had these? A: Yes, yes, of course. Yes now except, um, the 118 personnel …” And again; access records were held to be unnecessary [superflua] (pp 262-263): “…Attorney: do you know whether your presence was recorded? A: What do you mean recorded? …Attorney: Was you entry noted? Because I can’t find it anywhere? A: Our entry wasn’t noted because we went with the Public Minister. What am I supposed to do, make notes if there’s a Public Minister there?…But when there’s a Public Minister I don’t make notes and neither do my colleagues…“.

– Specific anticontamination clothing was not continually and correctly used, and this includes gloves, foot-covers, masks, and head covers. Even at the crime scene, in fact, there were persons present who wore only foot-covers and a pair of gloves, that is, they were in Meredith’s room normally dressed; and we emphasize again how 118 personnel entered without any precaution and even disturbed [movimentato] the crime scene (see the preceding transcription); there were also investigative personnel who were not correctly wearing masks and head protection.

– No indication was given of the correct use of disposable gloves, and in particular on whether they were changed at the moment of collection of each sample object.

–  Foot-covers were not changed inside the residence (see transcripts above), which gives rise to the possibility of a potential contamination. In this connection we emphasize what was explained above relative to the floor which: “…is largely a depository of evidence but at the same has the greatest contamination potential..”. We recall that the clasp was regularly observed and filmed at the very first inspection and remained on the floor for some 46 days, and how there were additional inspections and displacements of objects during this temporal hiatus”.

– The protocol during the collection of samples was not correct, i.e. failure to use disposable tweezers [pinzette], no preventive drying of biological traces, extraction on areas of low concentration, failure to take control samples, failure to wrap certain samples in paper, rushed sampling in open air [agitato campionamento nell’aria].

– The items were handled by more than one person without changing gloves, extracted without using appropriate tweezers (Deposition of Dr. Stefanoni, 10/4/2008, pp. 39-40); “…Q:…so the most you can imagine would be a movement of DNA already present on site? A: Yes…Q:…as regards then the reasoning you just gave, this also holds as a description of the change of hand which is of course always photographed or rather filmed…Why does this piece of fabric with the two clasps pass from the hand of one operator to the hand of another operator? A: But if the gloves are clean it absolutely doesn’t matter”.

– The initial position of discovery on the floor of the clasp was not the same after 46 days; and moreover it was touched several times in succession by several operators, after having been collected from the floor, and repositioned again on the floor; in disregard therefore of what is envisioned in Techniques of Crime Scene Investigation (Techniques of Crime Scene Investigation, Barry Fischer – CRC ed. 2003), that is: Accurately record the position of objects before removing them (caution: do not try to put the objects back in their original position).