Continuation Part Eight: Discussion of the Amanda Knox/Raffaele Sollecito case

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Strozzi said:
Boy was I excited. On a hunch, I went to the Italian version of EBay. I typed in ABI 7700 and 4 machines for sale came up. Ranging in price from $350 - $5,950. I searched for the seller's identity - hoping I would find one being sold used by "Stefanoni & Co." in Rome! :p.

Just my luck - all are being sold by US sellers.

See them on EBay Italiano at: http://www.ebay.it/sch/i.html?_trksid=p2050601.m570.l1313.TR0.TRC0.H0.Xabi+7700&_nkw=abi+7700&_sacat=0&_from=R40
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This is pretty good evidence that these are considered dinosaurs today. But that doesn't really surprise me since the software discussed in the manual is for a pretty old Macintosh. My guess is that ABI has a much more advanced model today.
 
Here is a document: Data Analysis on the ABI PRISM" 7700 Sequence Detection System: Setting Baselines and Thresholds, that may help some of you understand how the system is setup and the control the operator has. It also provides insight that there is much more information in the raw data file that the machine produces than what we can extract from the printouts.


One little part that I still find troubling is the Slope values. These values characterize the exponential growth of DNA in the cells during the run. A slope of -3.31 indicates that there is a 10 fold increase in DNA every 3.31 cycles. This is said to be 100% efficient because it is the same as doubling the DNA every cycle.

The way PCR works is that in each cycle the double strands of DNA are split apart and each half forms a new double. There is no possibility to do better than doubling every cycle (or 10 folds every 3.31 cycles). But in Stefinoni's lab the slowest growth was 10 fold in 3.31 cycles. All the others achieved a 10 fold growth in fewer than 3.31 cycles and a couple were as low as 2.5 cycles. This is strictly impossible.
 
Dan O. said:
Here is a document: Data Analysis on the ABI PRISM" 7700 Sequence Detection System: Setting Baselines and Thresholds, that may help some of you understand how the system is setup and the control the operator has. It also provides insight that there is much more information in the raw data file that the machine produces than what we can extract from the printouts.


One little part that I still find troubling is the Slope values. These values characterize the exponential growth of DNA in the cells during the run. A slope of -3.31 indicates that there is a 10 fold increase in DNA every 3.31 cycles. This is said to be 100% efficient because it is the same as doubling the DNA every cycle.

The way PCR works is that in each cycle the double strands of DNA are split apart and each half forms a new double. There is no possibility to do better than doubling every cycle (or 10 folds every 3.31 cycles). But in Stefinoni's lab the slowest growth was 10 fold in 3.31 cycles. All the others achieved a 10 fold growth in fewer than 3.31 cycles and a couple were as low as 2.5 cycles. This is strictly impossible.
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Are you sure of this???
 
acbytesla said:
Are you sure of this???
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I suppose they could have redefined math while I wasn't looking.

Why don't you ask Google what log10(1/2) is. Or show how you can fold a piece of paper 3 times and get 10 layers.

And why in hell have those printouts of the quantification data been poorly altered? It looks like someone cut out the dates and pasted in a different date. (Forget that. The original document is a spreadsheet that has been printed with the columns compressed to fit on an A4 page.)((But some of the "Last Modified Date"s are still questionable.))
 
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Here is an explanation of PCR efficiency that indicates it can be greater than 100%

http://www.lrgc.ca/Forms/qPCR-Handbook-flr.pdf
Efficiency
A PCR efficiency of 100% corresponds to a slope of –3.32, as determined by the following equation:
Efficiency = 10(-1/slope) –1
Ideally, the efficiency (E) of a PCR reaction should be 100%, meaning the tem- plate doubles after each cycle during exponential amplification. The actual efficiency can give valuable information about the reaction. Experimental fac- tors such as the length, secondary structure, and GC content of the amplicon can influence efficiency. Other conditions that may influence efficiency are the dynamics of the reaction itself, the use of non-optimal reagent concentrations, and enzyme quality, which can result in efficiencies below 90%. The presence of PCR inhibitors in one or more of the reagents can produce efficiencies of greater than 110%. A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope of between –3.58 and –3.10.​


Stefi's efficiencies range from a low of 101% to a high of 152%. All but 2 are above the 110% recommendation.
 
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Dan O. said:
I suppose they could have redefined math while I wasn't looking.

Why don't you ask Google what log10(1/2) is. Or show how you can fold a piece of paper 3 times and get 10 layers.

And why in hell have those printouts of the quantification data been poorly altered? It looks like someone cut out the dates and pasted in a different date. (Forget that. The original document is a spreadsheet that has been printed with the columns compressed to fit on an A4 page.)((But some of the "Last Modified Date"s are still questionable.))
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No that wasn't my question Dan. The question is did Stefanoni actually achieve a higher than a 100 percent efficiency or 3.31?
 
acbytesla said:
I keep reading about "abundant amount of DNA on the bra clasp but I haven't read how much that it is. I know the knife is beyond infinitesimal. Does this mean that the bra clasp is too?
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IIRC both the knife and bra clasp hook samples are LTN/LCN.

During the detention hearing Stefanoni called sample 36b (the knife blade sample) large. In fact she she was pressed to be more specific and then stated a couple of hundred picograms.

A year or so later during the trial we learn that this was a lie...(she claimed to have forgotten her notes during that earlier court testimony) and that this 36b sample was smaller...maybe 10 picograms...and like Planigale explains...below a certain level we can call this 0 (zero) picograms....especially back in 2007. So sample 36b which was consumed completely quantified as 10 picograms or maybe o picograms.

The bra clasp sample was larger...but still LTN/LCN and too small for Stefanonis lab to test. She was not equipped, not certified, not trained, and the machine manufacturer warned her that this testing was impossible and unreliable.

PS the kit was not designed to test LTN/LCN samples either.

And yet Stefanoni comes into court and presents what she has to know is incorrect data. And even worse this data has been reviewed and certified as reliable by the prosecution expert consultant...Biondo...who co-incidentally is Stefano's boss and head of her lab. Huh!

This is such a severe example of conflict of interest that I cant imagine the defense was not raising particular hell about it...and yet they did nothing, except sit on their hands.

You lie and I'll swear to it. Some sort of Italian judicial motto I think.
 
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Dan O. said:
Here is an explanation of PCR efficiency that indicates it can be greater than 100%

http://www.lrgc.ca/Forms/qPCR-Handbook-flr.pdf
Efficiency
A PCR efficiency of 100% corresponds to a slope of –3.32, as determined by the following equation:
Efficiency = 10(-1/slope) –1
Ideally, the efficiency (E) of a PCR reaction should be 100%, meaning the tem- plate doubles after each cycle during exponential amplification. The actual efficiency can give valuable information about the reaction. Experimental fac- tors such as the length, secondary structure, and GC content of the amplicon can influence efficiency. Other conditions that may influence efficiency are the dynamics of the reaction itself, the use of non-optimal reagent concentrations, and enzyme quality, which can result in efficiencies below 90%. The presence of PCR inhibitors in one or more of the reagents can produce efficiencies of greater than 110%. A good reaction should have an efficiency between 90% and 110%, which corresponds to a slope of between –3.58 and –3.10.​


Stefi's efficiencies range from a low of 101% to a high of 152%. All but 2 are above the 110% recommendation.
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What? This is very interesting. Which run was 152%?
 
Dan O. said:
That was the first run on the 7500.

Code: 
RUN_NR    Date	             Slope  Intercept  Fit  Efficiency
7500_017  5 Mar 08 12:45:59  -2.491648  28.8  0.923181  152%
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Lol. Every time someone takes a hard look at a piece of lab data, some other screwed up issue is found.

Hard to believe that people are supposed to spend basically the rest of their productive lives on jail based on this crap.
 
Does anyone know if Italy has FOI legislation. It would be interesting to put in a request around external QC, audit and accreditation assessments for the laboratory. I know the lab was not accredited at the time (they became accredited in 2009 - a response to this case?). But the initial assessment might give interesting insight. They will also now be required to keep records of contamination episodes, if they have contamination episodes now it would be hard to claim they did not then, but just failed to recognise / record them.

The single documented example we have of low level DNA contamination of a negative control is sufficient to prove low level DNA contamination affected Stefanoni's testing protocol and should in itself be sufficient to exclude LCN evidence.
 
RandyN said:
IIRC both the knife and bra clasp hook samples are LTN/LCN.

During the detention hearing Stefanoni called sample 36b (the knife blade sample) large. In fact she she was pressed to be more specific and then stated a couple of hundred picograms.

A year or so later during the trial we learn that this was a lie...(she claimed to have forgotten her notes during that earlier court testimony) and that this 36b sample was smaller...maybe 10 picograms...and like Planigale explains...below a certain level we can call this 0 (zero) picograms....especially back in 2007. So sample 36b which was consumed completely quantified as 10 picograms or maybe o picograms.
The bra clasp sample was larger...but still LTN/LCN and too small for Stefanonis lab to test. She was not equipped, not certified, not trained, and the machine manufacturer warned her that this testing was impossible and unreliable.

PS the kit was not designed to test LTN/LCN samples either.

And yet Stefanoni comes into court and presents what she has to know is incorrect data. And even worse this data has been reviewed and certified as reliable by the prosecution expert consultant...Biondo...who co-incidentally is Stefano's boss and head of her lab. Huh!

This is such a severe example of conflict of interest that I cant imagine the defense was not raising particular hell about it...and yet they did nothing, except sit on their hands.

You lie and I'll swear to it. Some sort of Italian judicial motto I think.
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Can we please get this right? Just once? The blade sample weighed zero tons.
 
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does the corruption of the evidence have any effect on the DNA evidence on Rudy?
Granted, the fingerprint evidence already seems pretty solid.
 
Desert Fox said:
does the corruption of the evidence have any effect on the DNA evidence on Rudy?
Granted, the fingerprint evidence already seems pretty solid.
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In a proper jurisdiction I think it would all have to be thrown out and done again - if that were possible. Just like the case Kaosium used to reference of an FBI lab person who by-passed a particular stage in the sequence in about 200 cases. All her work had to be re-done and her lapses were nothing like Stefanoni's.
 
anglolawyer said:
Can we please get this right? Just once? The blade sample weighed zero tons.
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Long ton or short ton, or (given that you Limeys are always adding unnecessary letters to God-fearing, perfectly good 'Merican words) a tonne?

Go to Gaol, do no pass Go.....
 
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Diocletus said:
Dan, since you're good at math, this seems pertinent to the baseline and efficiency issues:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665230/
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The current study shows how an improper baseline setting severely affects the estimated PCR efficiencies and will thus increase the variability as well as the bias in the reported absolute and relative levels of gene expression.​

an overestimation of the baseline leads to an overestimation of the PCR efficiency​

The baseline represents a constant fluorescence that exists before the amplification of the DNA. If this is not measured correctly it throws all of the numbers off and can result in order of magnitude errors in the estimates of the starting DNA quantities.
 
Dan O. said:
The current study shows how an improper baseline setting severely affects the estimated PCR efficiencies and will thus increase the variability as well as the bias in the reported absolute and relative levels of gene expression.​

an overestimation of the baseline leads to an overestimation of the PCR efficiency​

The baseline represents a constant fluorescence that exists before the amplification of the DNA. If this is not measured correctly it throws all of the numbers off and can result in order of magnitude errors in the estimates of the starting DNA quantities.
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Yup, and the baselines for 544 and 570 are relatively high. And, she abandoned the Real Time machine after she analyzed each of these runs. So, what does that tell us about what she thought about the reliability of the quantification data for, e.g., 165b?

And, why was her baseline jumping up by a magnitude of 30%?
 
So what we have is a host of different nonsense motives.
No prints, no hair, no fibers.
Even if the DNA evidence was valid and was not due to lab contamination, there are perfectly reasonable ways that the DNA evidence could have occurred.
However, all the DNA evidence now appears to be completely invalid.

So, what is the evidence of guilt. . . . Psychic determination :confused: :boggled:
 
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