Seminal Fluid & The Failed Sexual Assault Investigation


The Rape Kit Results

The police understood immediately that the case involved a sexual assault. Accordingly, the examining pathologist, Dr. Lalli, performed a rape kit, i.e., a set of procedures used to collect forensic evidence in a sexual assault case. This resulted in the collection of vaginal and rectal swabs from the victim, in the hope that the swabs could be analyzed to reveal the DNA of the attacker.

The vaginal (Exhibit 12) and rectal (Exhibit 13) swabs were among the first items analyzed by the Scientific Police upon delivery to the laboratory in Rome, on or about November 5, 2007. Two traces of presumed seminal fluid were isolated and extracted from each swab, resulting in trace nos. 12a and 12b, and 13a and 13b.

The “a” traces were then subjected to a fractionation procedure, by which sperm and nonsperm DNA were separated. The so-called “nonsperm” fraction (i.e., the part of the trace containing no sperm DNA) were designated as subtrace “a1”, and the so-called “sperm” fractions (i.e., containing the isolated sperm cells) were designated as subtrace “a2”.

The “b” traces were not subjected fractionation, but instead, were subjected to standard STR analysis, and in the case of the vaginal swab, to Y-chromosome STR analysis (curiously, there is no indication that a Y-chromosome STR analysis was performed for the rectal swab).

Early Exclusion of Sollecito and Lumumba as the Attacker

Critically, the Y-chromosome STR analysis of the vaginal swab returned a male profile (that is now known to correspond to Rudy Guede). This result was obtained on or about November 6, 2007, and while it may not have been known at the time that the profile belonged to Rudy Guede, it was known that the profile did not match the reference profiles for either Raffaele Sollecito or Patrick Lumumba. Thus, Sollecito and Lumumba were excluded as the attackers by about November 6, 2007. This exculpatory information, however, was not disclosed to the court or the defense prior to or during the pre-trial detention hearing on November 8, 2007.

Missing Sperm-Specific Analyses

The laboratory records also reveal that both of the “sperm fractions,” i.e., the subtraces that were supposed to contain only sperm, tested positive for human DNA. Although the amounts of detected DNA were relatively small (1200 and 700 picograms, respectively), the quantities were well within the parameters that the lab was using to determine whether to further analyze the samples. Indeed, discernable gaps in the lab records suggest that these DNA-positive sperm fractions were in fact subjected to genetic profiling as profile nos. 626 and 628. However, no amplification or electrophoresis records corresponding to profile nos. 626 and 628 were ever disclosed by the prosecution. As shown in the video below, crime scene investigator/lab technician Patrizia Stefanoni can be heard on November 3 telling her assistant of an urgent need to test “presumed seminal fluid”. They knew the perpetrator had ejaculated. Stefanoni would later claim testing the stain would compromise the shoe print evidence.

The Semen Stain

In addition to the swabs collected by the pathologist, Dr. Lalli, there was a semen stain directly between the victim’s legs on the pillow Guede positioned her body on. The stain was only discovered by defense expert Professor Vinci when he was finally able to examine the pillowcase May 25th, 2009. Incredibly, the prosecution and Kercher family attorney, Francesco Maresca, resisted requests to test the semen stain and no court has allowed it to be tested. As with the sperm fraction samples from Dr. Lalli’s swabs, the identity of the semen stain donor remains unverified by any disclosed genetic testing.

Possible Freudian slip by lead detective Monica Napoleoni?

Lead Detective Monica Napoleoni testified Guede’s DNA was found on the pillow yet according to Patrizia Stefanoni it wasn’t tested. (p25 Transcript February 28, 2009)

Question: Listen do you know the results from Scientific Police, more or less obviously, not in detail because then we’ll ask them. I wanted to ask you if you remember on which exhibits the DNA of Rudy Guede was found
Answer: Well, for sure the DNA of Rudy Guede, it was found on the pillow that was under the victim, on the toilet paper with the feces in the bathroom and on the vaginal swab but I don’t know if it was DNA however they call it Y-chromosome and I’m not a biologist, I’m not able to report on this.

The Injuries

From pathologist Dr Luca Lalli’s preliminary report: (Ricciarelli Report November 30, 2007 p4)

In such circumstance it had been found that the labia majora, the interlabial sulci, the external side of the labia minora, the clitoris, the periclitorial and urethral regions did not present any kind of trauma. The hymen presented proof of previous sexual relationships. On the inner side of the labia minora had been observed purple ecchymotic lesions, both on the left and right sides. In the circumstance it had been used a high vaginal swab. On the vaginal fornix it had been found and sampled a dense material of dark yellow color.

In the anal and perianal region it had been found that the anal sphincter was expanded by 2-3 centimeters and that a dense material beyond the anal contour was present. On the anorectal line were present small discontinuities of the mucosa of dubious origin. Posteriorly were present small ecchymotic purple colored lesions.

The injuries were covered extensively by both prosecution and defense experts at the preliminary hearing and trial but notably prosecution expert, Professor Mauro Bacci, said there was anal penetration and no evidence of constipation to explain the anal dilation of 2-3cm (Transcript April 19, 2008 p68). Professor Bacci testified you see anal dilation naturally in children post-mortem but not adults. (Transcript April 19, 2009 starting p18)

Massei Report page 121

Professor Bacci, a consultant appointed by the Public Prosecutor together with Professor Marchionni and Dr. Liviero, gave his assessment at the hearing on April 18, 2009.


Professor Bacci also considered it possible that there was violence and anal type penetration, highlighting the presence of small pinpoint haemorrhages, small bruises that other consultants attributed to constipation: he was not fully convinced by this interpretation, since there was no evidence of such constipation, which would have been a very important issue.

Professor Vinci’s Report

Sollecito defense expert, Professsor Francesco Vinci, presented the results of his analysis of the pillow case in the following slide presentation and below is an English translation of his report:

Amanda Knox case

Homicide of Merdith Kercher
Tribune of Perugia

Additional personal observations regarding the pillow case
of the pillow found below the body of the deceased

Prof. Francesco Vinci
Professor of Legal Medicine, Forensic Pathology,
Crime Scene Investigation and Criminal Techniques
University of Studies of Bari
Section of Legal Medicine (Di.M.I.M.P.)

The Homicide of Meredith Kercher- Tribune of Perugia- Considerations of Prof. Francesco Vinci- University of Studies of Bari


TOOLS USED – professional digital reflex camera
– computerized system for image analysis
– graphics software
– alternative illumination system (Crimescope)
Top (A) Bottom (B)

One notes two distinct traces apparently of the same aspect different from the obvious signs of blood

Top (A)

This enlargement better shows the characteristics of the two stains in question

The examination with the Crimescope shows the intense fluorescence of the stains

Filter 475- bandwidth 45 nm

The presence of this thin segment which connects the two stains is very notable

The enlargement of the image (showing the relief and formations of the edges) clearly shows the presence of hidden characteristics of the stain

The further processing of the image (delineating border areas and assigning colors) shows that the stains can be clearly distinguished by five zones

These elements lead us to believe that in fact it is through five distinct moments of affixing of the substance that produced the stains: stain 2 is in fact over-laying 1 and 5 is over-laying 4.

At this point: What is the nature of the
substance which is creating the stain?

It is evident that a certain answer can only be derived by carrying out a laboratory test.

But we can formulate an hypothesis based on observation.

Compendium of the characteristics of the observed stains:
– limited dimensions: Max cm 3×2;
– color observed by eye: white-yellow;
– irregular and jagged edges (like on a geographical map);
– intense fluorescence with observation with Crimescope;

All of these are strongly indicative of sperm.

4. Sperm
In certain cases of carnal rape or libidinous acts it can be necessary to compute tests for the presumed traces of sperm.
On the garments or other permeable fabrics sperm assumes the aspect of a whitish-yellow stain, denser and darker at the edges, with sinuous borders (like a geographical map); on objects or non-permeable fabrics dried sperm presents itself instead in the form of very light grey scales, with little adhesion. With light (ultraviolet) of Wood (produced by use of a mercury vapor lamp, using a filtration with oxidized nickel with glass), the stains show a bluish fluorescence, which allows a quick orientation: especially when they are small or situated on a dirty fabric or on a color similar to them.
For a more precise diagnosis one must however proceed with further analysis, by using other methods.

The interpretation of this segment (with respect to the density of the liquid) gives us another element which confirms the diagnosis of sperm:

General physical characteristics
Immediately after emission the spermatic fluid coagulates, but with a gelatinous consistency and with a milky whitish color; it also has a characteristic odor which comes from the oxidization of the sperm cells. Within a few minutes of emission, the process of dissolution occurs from the coagulation and refluidification is completed generally within 20-30 minutes by means of the prostatic seminal plasma enzymes which split the seminal plasma and together with numerous other enzymes which are: lysozyme, alphachimotripsin, hyalurondinase, etc…
Sperm thus acquires a translucent aspect more or less opalescent depending on, largely, the cellular contents. After the complete liquefaction it is possible to give a value to the viscosity of the liquid, by putting into a test tube: sperm at normal viscosity coagulates slowly, drop by drop, the formation of filaments indicates an augmented viscosity. The notable augmentation of the viscosity is apparently due to an enzymatic process which involves the fibrinolysin and the fibrinogenase and infers more than anything nemaspermatic mobility. A diminution of viscosity, with the watery aspect of seminal plasma, is accompanied on the other hand, very often with oligospermia.


1) What time do the the stains date back to?

The stains from our objective point of view can no longer be reliably dated by any method, nor by the means of elaboration by the laboratory tests conducted.

Evidence to that effect would have had to have been made by an examination carried out during the time of the discovery of the corpse, by analyzing the degree humidity of the stain.

In order to measure the degree of humidity of the stain one would have used a simple hygrometer for cloth

Of course, this is based on the assumption that the spots were already identified during the initial and early stages of the inspection. The occurrence of this in this case is more than clear given that the stains were enhanced with the Crimescope which were more than visible to the naked eye.

2) The affixing of the spots is directly derived from an ejaculation or is due to leaking of semen from elsewhere?For a useful deduction in that sense it is necessary to make a few preliminary considerations based on the circumstances of the discovery of the body

From these images the stains in question are clearly shown situated exactly in front of the genital region of Kercher

In any case we do not believe that the highlighted traces are related to material coming out from the vagina of Kercher (where genetic material belonging to Rudy Guede was also found), perhaps after death.In fact in this case it would not keep the viscosity necessary to show the filament attributed in zone 3 of the tracks

Brief notes on the physiology of male ejaculation

Ejaculation (the mecchanism which allows the exiting of sperm through the urethra of the penis) can be subdivided in two phases:
The first phase, i.e. the emission, takes place through the contraction of the internal organs and comes immediately before the actual ejaculation. In other words , this contraction of the interior apparatus, brings together the various components of semen ( sperm and the prostate fluid) and transfers the liquid to the bulbar urethra immediately before orgasm, to allow the mechanism to expel externally ejaculatory sperm.
The root of the penis, where it engages in the pelvic floor, is surrounded by strong striated muscles which wrap around this part of the penis. During the ejaculatory orgasm, there is a series of involuntary contractions of these muscles.
It is exactly these contractions that represent the drive motor of the ejaculation, squeezing the root of the penis and urethra, causing the expulsion of the semen in successive jets and in progressively lower quantities.

Taking into account these elementary notions of the physiology of ejaculation we justifiably note that the stains in question are in exact accordance with this mechanism.

In fact, this view is supported by:
– The overlapping of the spots;
– The different sizes of these.

In accordance with the decreasing size of the spots, it could also be envisaged that they were affixed in the following order:


Therefore in Conclusion:

– The spots we identified are very likely made of sperm;
– It is not possible to state the dating of the tracks;
– Ejaculation occurred outside of the vagina of Kercher;
– It is possible still today to verify the nature of the traces in question in the laboratory (by means of DNA Research)

The observation of the pillowcase under court sequestration using the Crimescope lighting system, highlighted another important finding:

One can see very luminescent traces, which are absolutely not visible to the naked eye

You can see more clearly the tracks we have highlighted

The enlargement shows with greater clarity the other traces revealed through the observation using Crimescope

The morphology of these traces (shown with the Crimescope), show with sufficient definition a particularity of the plantar part of the sole of the left shoe of the Nike model Outbreak 2 size 44.5 which we used for more tests. The overlaying of the traces with the sole of the shoe is strongly demonstrative with regard to:

This track (highlighted with Crimescope), by integrating our consultation on the shoe prints on the pillow) must therefore be placed in relation to one of the fifth placements (footprint n.3) of a plantare portion of part of a sole of a left shoe, Nike brand model Outbreak 2.

Shoeprint no. 2
Shoeprint no. 3
Shoeprint no. 1

Thank you for your attention


Case Files

RTIGF Report
Test results index translated to English
Stato Avanzamento Lavori (SAL)


Amanda Knox retrial: judge allows new DNA test on alleged murder weapon – “But, in a blow for Knox and Sollecito, the court rejected almost all their lawyers’ requests for further evidence or testimony. For instance, the court said it would not analyse semen stains at the crime scene.”

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