1. The Lab’s Records Prove Contamination
It is simple to find the contaminated negative control in Batch 4. Stefanoni’s own quantification results for Real Time run no. 564 show the negative control (NTC, or No Template Control) located at wells B10-12, and well B11 shows a “Ct” value of 34.30. (Quantificazione p. 14)
As explained below, this Ct result for Well B11 shows that the negative control is contaminated. Moreover, in the Fast Time PCR Run dated March 7, 2008, there is a similar occurrence of a positive Ct value in a negative control, which demonstrates that PCR contamination in Stefanoni’s lab is not an isolated occurrence. See Quantificazione.pdf (p. 29/47, Wells B10 and B12).
The abbreviation “Ct” stands for “Cycle Threshold.” Simply put, a PCR reaction consists of a number of cycles of DNA amplification, each of which is supposed to increase the quantity of DNA in the reaction. The Ct value is the measurement of the number of cycles of amplification that have occurred at the point at which a critical quantity of DNA is detected in the reaction. Simply put, a Ct of 34.30 means that the critical quantity of DNA was detected during the 34th cycle of amplification. Since the lab ran each sample through 50 cycles of amplification, a negative result would show a Ct of 50 (meaning that the 50th cycle was completed and the critical quantity of DNA was never found), as can be seen in Wells B10 and B12. Well B11 is “positive” for contamination because it detected DNA in the 34th cycle.
2. The Lab Contamination is Low-Quantity, Random, and Comparable to 36b
The approximate amount of DNA that is contained in the contaminated negative control, B11, can be extrapolated to be about 75 picograms (in 50 uL). Generally, the quantity of DNA within a sample is proportionate to the Ct value. Although no quantity information is printed for the negative controls, including Well B11, Well D9 shows a Ct of 33.32 which corresponds to a quantity of 1.9 pg/uL; and, Well E4 shows a Ct of 37.15 corresponding to a quantity of .1 pg/uL. Therefore, extrapolation shows that B11, with a Ct of 34.30, has a concentration of about 1.5 pg/uL, which would yield about 75 picograms of DNA in the entire 50 uL template.
The fact that contamination has been shown to have occurred in a PCR process in Stefanoni’s Lab puts in question her methods and the value of her results, particularly her LCN results, which are comparable to the results of the contaminated control. In fact, the kind of contamination that has occurred here (PCR contamination demonstrated by a contaminated negative control) is random contamination, likely caused by “carryover” in the sample loading process or airborne contaminants in the PCR processing area. Indeed, the manual for Stefanoni’s own equipment describes this kind of contamination as follows:
In this situation, NTC contamination randomly occurs when loading the DNA templates into the PCR plate. If the contamination is due to plate loading, the NTC will show amplification in some or all of the NTCs at varying CT values.
- Use clean working practices to avoid template contamination.
- To avoid contamination from previous PCRs, incorporate an AmpErase Uracil N-Glycosylase (UNG) or Uracil-DNA Glycosylase (UDG) step prior to PCR to reduce carryover contamination.
- For optimal PCR results, Applied Biosystems recommends that you use separate working areas for PCR mix preparation, template addition, and performing the PCR reactions.
Unfortunately, it does not appear that Patrizia Stefanoni used the requisite “clean working practices” or followed any other remedial recommendations, as demonstrated to the subsequent repeat (i.e., on March 7, 2008) of a contaminated negative control in her real time PCR negative controls. Since this PCR contamination occurred, was repeated, and was random, PCR contamination could have occurred at any time, including in the STR amplification process that yielded the allegedly “incriminating” electropherograms for Rep. 36b and Rep. 165; in short, the electropherograms for Rep. 36b and Rep. 165 could reflect the results of laboratory contamination instead of DNA actually extracted from the exhibits.
It is important to note that the amount of DNA detected in the contaminated negative control is comparable to the result for sample 36b. For example, although Stefanoni used a nonspecific method of quantification for Rep. 36b, the strength of the RFU scale for 36b is weaker than the RFU scale for Egram No. 950, a 35 picogram sample. This suggests that 36b contained less than 35 picograms of DNA. In comparison, the contaminated negative control shows a quantity of about 75 picograms. Thus, Rep. 36b actually contains less DNA than can be seen in a known product of contamination (i.e. well B11), which of course, suggests that the DNA in 36b easily could also be the result of contamination.
3. Contrary to Her Testimony, Stefanoni Knew of the Contamination
Stefanoni’s quantification records also show that she was aware of, and reacted to, the contamination of a negative control in Batch 4. As established in the Batch 1 through 4 analyses posted here The Prosecution’s Massive Suppression of Lab Data and below, Stefanoni’s practice was to consider a quantification to be positive, and to submit the respective sample to STR amplification, if the sample achieved quantification with a Ct below 37. Yet, in RT-qPCR Run No. 564, we see a sample (Well D9-10) that returned a Ct 32.9 (average between two wells), which was NOT submitted to amplification, and further, is identified in Stefanoni’s Report as “negative” for quantification.
It is unprecedented, as demonstrated in the charts for batches 1 through 4, that Stefanoni would treat a Ct of 32.9 as quantification-negative, and fail to submit such a sample to subsequent STR amplification (see, e.g., Egram No. 950 (Egram for sample with Ct=35)). The simple explanation for this deviation from her standard practice appears to be that Stefanoni was cognizant that a negative control had shown contamination at Ct=34.30, and she therefore deemed Well D9-10, returning a Ct of 32.9, to be a result or possible result of contamination due to the closeness (+/- 1.4) of its Ct result (32.9) with the Ct result for the contaminated negative control (Ct 34.30). The response, of course, was not to disclose the contamination or to repeat the PCR process; but rather, simply to disregard the sample that she thought looked too much like contamination.
Moreover, although it can be extrapolated that the contaminated negative control was further analyzed as STR amplification ID No. 452 (see chart), the results of this amplification have not been disclosed, and therefore, the resulting profile has not been inspected to determine whether it is the profile of Kercher, Sollecito or some other person.
4. The Coverup
As noted above, Stefanoni was aware of instances of actual laboratory contamination. However, at trial, these instances were actively covered-up through false and misleading testimony on the issue of contamination . This cover-up duped the courts into concluding that Stefanoni’s laboratory was pristine and error-free.
On May 23, 2009 (pp 54-55) Stefanoni testified as follows:
PM Mignini: Listen, how many years have you been doing this work?
Stefanoni Nearly 7 years.
PM Mignini: Approximately 7 years. Have you worked on cases similar to this?
Stefanoni Yes. Yes, yes.
PM Mignini: Many similar cases?
Stefanoni: Yes, various cases of similar complexity, basically, yes.
PM Mignini: Do you remember if you’ve always followed the same method?
PM Mignini: In the way you basically collected the samples and then in the analysis?
PM Mignini: Right, do you remember any confirmed contamination of samples?
Stefanoni: No, such a problem has never been highlighted to me.
PM Mignini: So you have absolutely no recollection [of such a thing]… and you have always followed this same method that you have described to us today?
This misleading testimony was picked up by Judge Massei, who in support of his conclusion that there was no laboratory contamination, noted:
In response to a specific question on this point, Dr. Stefanoni declared that she had been working as a biologist for seven years, had always used the same methodology, and had never heard that any problem of contamination of exhibits had occurred.” (Massei p. 220).
5. Batch 4 (13-18 December 2007)
- Batch 4 contains 72 amplifications, 23 of which are missing, for a suppression rate of 32%.
- The missing profiles consist of: negative controls (3); items that appear to have been collected from Rudy’s Guede’s apartment (14 samples), samples from Sollecito’s shoes (4); 2 samples of Kercher’s blood, and, curiously, 1 sample of presumed “saliva” from the kitchen knife (Rep. 36).
- Critically, one of the Negative Controls for this batch shows contamination: see No Template Control located at position B10-12 of RT-qPCR Run No. 564.
- There are two sets of field-numbering (A to E and 1 to 12) for the samples collected from Guede’s apartment, attesting to the two separate searches of Guede’s apartment (on November 16- and November 20). The police seized a shoe box (presumably for Nike Outbreaks), a towel, toothbrush, sink strainer, jeans and a museum ticket (Items A through E). Curiously, none of the extraction or quantification records for the initial testing of any of the Guede items have been produced, and it is only from the Rep. Nos. (i.e., 58 and 148-163) and a Plate No. (i.e., 403) that we can estimate that the toothbrush, Rep. No. 58, was first tested around November 16, and the rest of the Guede items were analyzed on or shortly before December 13, 2007.
Selected DNA results
Polizia Scientifica RTIGF Report
Test results index translated to English
Stato Avanzamento Lavori (SAL)
DNA profiles for Guede, Kercher, Sollecito, and Knox
DNA evidence/test results and e-grams for knife