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

[acbytesla]

As for the 'too lows' being more than six, she didn't just use the Qubit on the knife as I understand, I'd need to see the rest of the page that Randy (W--surfer) recently posted from Frank Sfarzo to see what the rest of them are for.

The Qubit is not at all suited for low template work, it's detection level isn't high enough. Remember that with many DNA samples there will be plenty (relatively) of material to take a sample of. Someone who has bleed out might well have liters of blood on the floor with gobs and gobs (relatively) of DNA in it. Thus one takes a small bit of that and gets a sample and puts part of that through the Qubit and gets a value per microliter that amounts to having 10 nanograms (total) in the tube. As most of those machines are designed for samples of about one nanogram you'd then split your sample into tenths and have ten one nanogram sized samples to amplify. That's one of the main reasons for the quantification step, most of the machines and software are designed for samples of 0.5-1.5 nanograms. Too much and it may register off the chart, too little and there may be difficulty interpreting the results.

Isn't this a good rationale then for Stefanoni to test all the 36 samples with the Real time after? But then it makes you wonder why Stefanoni tested any of the samples with the Qubit?

 

[Kaosium]

Then why does the machine's printout print results in a normal format, instead of printing WARNING, THESE RESULTS MAY BE FALSE. GARBAGE IN, GARBAGE OUT! . These machines are sold to police forensic labs and it is foreseeable that the printouts will be used to pull the woll over the eyes of ordinary people. Why is the disclaimer not printed in the data output? It is important enough to include in the operating manuals; why not in the printout?

Because the reality is that you can get a good profile from under 150 RFUs, the difficulty in interpretation becomes where that DNA came from. Objectively, the most likely place for that DNA to come from is in fact the lab that tested dozens if not hundreds of items with DNA from the subject on them, or from the handling of that item by someone who was also handling items with that subjects DNA on them. That's the reason for all those stringent protocols requiring labs that are beyond sterile and the changing of gloves and so forth, to minimize the possibility the DNA came from the (otherwise) more likely sources and was actually on the item at the moment the crime was committed. The lower you go the more of a danger this would be, those low levels of contamination that can be claimed are by labs who generally would throw this crap out as being below the minimum threshold.

 

[Skind]

IIRC in the "Math on Trial" thread, Leila said that Stefanoni marked the quantitation of 36B as positive long after the quantitation experiment, because she got an egram. If this is an accurate summary, then it is an object lesson in how not to keep laboratory records. You are supposed to write an up-to-the-minute summary in your lab notebook, so that later thoughts do not color your observations.

Essentially, Leila is accussing Stefanoni of fabricating official documents.

 

[Strozzi]

The problem to a certain degree Strozzi is that it takes multiple machines to get a result. For example. Stefanoni tests 36B for DNA gets a negative result with the Qubit then subjects 36B to the Speedvac to concentrate the DNA and then runs it through electropherisis. It's not the manufacturer's fault that the tech misused the equipment and the Genemapper software has no idea if there was "garbage in".

So it is the fault of the system integrator! Sue them, too!

 

[Kaosium]

IIRC in the "Math on Trial" thread, Leila said that Stefanoni marked the quantitation of 36B as positive long after the quantitation experiment, because she got an egram. If this is an accurate summary, then it is an object lesson in how not to keep laboratory records. You are supposed to write an up-to-the-minute summary in your lab notebook, so that later thoughts do not color your observations.

Thanks, that makes more sense and was something I'd wondered about.

Now, if I recall correctly the rest of the knifes samples (D-G) were run well after the knife had been presented to the world as 'the murder knife.'

If you thought you had a (real) positive result in a murder case from one sample, how long would it take you to run the rest of the samples if the one you thought 'positive' was registering so low as to be below the scientific standards (then) in place anywhere in the world? Wouldn't that be the first thing you would do? Maybe see if those other samples which registered the same exact reading with the Qubit might also have the DNA you're looking for in them?


I looked it up, those other samples weren't run until December 18th, 2007:

It should be emphasized that from an examination of the Real Time PCR reports exhibited, it appears that quantification using this method was only carried out, on 18 December 2007, on the DNA extracts indicated with the following numbers:

48649 = sample D

48651 = sample E

48654 = sample F

48655 = sample G

 

[Diocletus]

As for the 'too lows' being more than six, she didn't just use the Qubit on the knife as I understand, I'd need to see the rest of the page that Randy (W--surfer) recently posted from Frank Sfarzo to see what the rest of them are for.

Or you could just go here:

http://murderofmeredithkercher.com/batch-two-kitchen-knife-36b-suppression-90-profiles/

Also, see the end of this: http://murderofmeredithkercher.com/...en-samples-early-exclusion-sollecito-lumumba/

 

[Diocletus]

I almost chimed in on this when you brought it up, but you're out of luck. Those machines come with disclaimers and in fact one of them (or it may have been the Genemapper software) says that it's not recommended to attempt analyzing anything below 150 RFUs!

Apparently the disclaimer should be written in Italian.

 

[Chris_Halkides]

more on PCR and fluorescence

Thanks Chris. Mitosis is where cells split and double, right? But PCR doesn't double the number of cells in the same way with each cycle? It just doubles the number of DNA strands without cellular division? I'm really curious how the Qubit works. I'm perusing the manual now..but the manual seems to be for the Qubit 2.0. I'm guessing that Stefanoni was using the 1.0 version of this machine? So I'm wondering if the difference between the two is significantly different?

acbytesla,

PCR is a cell-free (in vitro) means of doubling the amount of DNA per cycle. Links to basics of real-time PCR here and here. From the first link: "The Taqman probe is an oligonucleotide that contains a fluorescent reporter dye bound to the 5' end and a quencher on the 3' end. The probe is designed to bind to the target DNA sequence. While the dye and quencher are intact, there is no fluorescence. When the polymerase elongates, it is able to cleave the probe, separate the reporter from its quencher, and fluoresce. This fluorescent signal is captured by Real-Time PCR." IIRC DNA polymerase uses its exonuclease activity to cleave the probe.

The Qubit uses a dye which preferentially binds to double stranded DNA. This link is not specifically about the Qubit, but it is helpful.

 

[RandyN]

As for the 'too lows' being more than six, she didn't just use the Qubit on the knife as I understand, I'd need to see the rest of the page that Randy (W--surfer) recently posted from Frank Sfarzo to see what the rest of them are for.

The Qubit is not at all suited for low template work, it's detection level doesn't go low enough. Remember that with many DNA samples there will be plenty (relatively) of material to take a sample of. Someone who has bleed out might well have liters of blood on the floor with gobs and gobs (relatively) of DNA in it. Thus one takes a small bit of that and gets a sample and puts part of that through the Qubit and gets a value per microliter that amounts to having 10 nanograms (total) in the tube. As most of those machines are designed for samples of about one nanogram you'd then split your sample into tenths and have ten one nanogram sized samples to amplify. That's one of the main reasons for the quantification step, most of the machines and software are designed for samples of 0.5-1.5 nanograms. Too much and it may register off the chart, too little and there may be difficulty interpreting the results.

This is what I believe is at the heart of the lab work deception. Stefanoni doesn't want conclusive proof...she wants a "shadow of doubt" to appear to be possible. In fact Mignini bases his whole case on exactly this type of deception.

The Qubit is tossed in so as to create confusion and avoid the EDF record trap she would be in if she just processed it all on the one machine.

It is notable that Stefanoni was unqualified to do LTN/LCN work. Her lab was neither certified or equipped to do LTN/LCN work. And especially in 2007 the scientific community voiced opinion against using such suspicious and small data samples in criminal cases at all. (They still do not recommend it)

Plus it is now confirmed that control sample data has been manipulated and or withheld so as to allow the impression that there never was contamination...when in fact the data proves exactly that there WAS CONTAMINATION! And the scientist went into court and lied about quantification method and size and the reasoning was never explained.

So why B and not C or D etc? She can never answer that. But did a lawyer bother to ask her? Even C and V pussy footed around the science giving this lab the chance to save face and cut and run...but they remained stupid and thought they could simply have Comodi state "we give what we feel is necessary". Right! Well so far these defense lawyers have allowed as much.

Besides the jointly signed letter of several scientists (from USA?) who all indicated scientific irregularity there were few real professionals in Italy who stood up against this shoddy unprofessional and likely highly corrupt data. It is not just the one sample...but rather volumes of wrong...chain of custody error, mishandling error, improper prep and storage error, and improper testing and data collection and data discovery. Probably not error but rather a deliberate effort to deceive.

If it was one or 5 things ....well maybe. But its hundreds.

Sue the machine manufacturer? Sure absolutely. Even if you lose you still force them to pull their heads out of the sand and explain why they are not at fault. And in turn shine the glaring light of blame where it belongs...on Italy.

And forget the disclaimers. They were obviously not good enough to prevent this gross misuse of their equipment by friggin pretend Italian doctors. Even Novelli must have been confused by the limitations going by his testimony.

So if not the Italians then who? The manufacturer certainly. Someone is wrong...all of Italian science or the guys making making these easily manipulated machines. The warning must not be strong enough to overcome a whole countries scientific community...or perhaps the Italian scientists are all deaf mutes? You decide.

 

[acbytesla]

acbytesla,

PCR is a cell-free (in vitro) means of doubling the amount of DNA per cycle. Links to basics of real-time PCR here and here. From the first link: "The Taqman probe is an oligonucleotide that contains a fluorescent reporter dye bound to the 5' end and a quencher on the 3' end. The probe is designed to bind to the target DNA sequence. While the dye and quencher are intact, there is no fluorescence. When the polymerase elongates, it is able to cleave the probe, separate the reporter from its quencher, and fluoresce. This fluorescent signal is captured by Real-Time PCR." IIRC DNA polymerase uses its exonuclease activity to cleave the probe.

The Qubit uses a dye which preferentially binds to double stranded DNA. This link is not specifically about the Qubit, but it is helpful.

Thank you for the info. So the Qubit DOESN'T amplify the sample at all? It only tells us if there is any DNA to begin with?

 

[Kaosium]

Or you could just go here:

http://murderofmeredithkercher.com/batch-two-kitchen-knife-36b-suppression-90-profiles/

Also, see the end of this: http://murderofmeredithkercher.com/...en-samples-early-exclusion-sollecito-lumumba/

3. 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.

Gott im Himmel!

I assume the page is not finished as I cannot find a link to that video thus I'm guessing it hasn't been linked yet. Or I'm just blind!

This is just staggering. To think that Maresca participated in this travesty, running interference as Mignini tried to take the lives of two innocents to preserve the fantasy Mignini created which minimized the role of the man who raped and murdered the daughter of his clients.

The cat blood is interesting, I was not aware that there were so many traces that registered human DNA, my initial thought was perhaps human DNA had gotten into cat blood, but the totality of the evidence suggests that's unlikely. Has anyone put together a map of where this all is, as in where the DNA and blood stains were found? I've a mental map of the cottage and where everything fits there, but it's a void on the outside except for the window of course.

Here's to you and everyone involved responsible for this!


I love that scene, nothing quite like solving a problem with the proper application of a gigajoule or two!

 

[JREF2010]

So why B and not C or D etc? She can never answer that. But did a lawyer bother to ask her? Even C and V pussy footed around the science giving this lab the chance to save face and cut and run...but they remained stupid and thought they could simply have Comodi state "we give what we feel is necessary". Right! Well so far these defense lawyers have allowed as much.
.

Like Hellman regrets not stating the interrogation was corrupt, C&V possibly did what you said, to give the "system/Stephony" a chance to save face(in other words C&V pussy foot around the truth) and maybe C&V even had a little fear of retaliation from the Nazi like Italian inbred legal politics pressing false charges. This is a place that can throw you in prison for a year without charges after all.

Who will investigate Stephony?
Will Toto's prison death ever be investigated? Probably not, but I bet Frank Sfarzo has a story of Toto's odd-timing and odd location of Totos last days.
Frank was writing on that very subject, abuse and death in the prisons, shortly before Migninni convinced some Florentine Judges to remove his website Perugia Shock. Stephony is part of the tainted-system.
Stephony represents Italys system as a cess-pool of corruption, imo.

I used to struggle with this Forensic work, trying to decide if Stephony & crew were ignorant or corrupt, until I finally realized they were both.

 

[Strozzi]

Kaohsiung comment
. . . I looked it up, those other samples weren't run until December 18th, 2007:

Originally Posted by C&V
It should be emphasized that from an examination of the Real Time PCR reports exhibited, it appears that quantification using this method was only carried out, on 18 December 2007, on the DNA extracts indicated with the following numbers:

48649 = sample D

48651 = sample E

48654 = sample F

48655 = sample G
__________________
"Honi soit qui mal y pense."


Strozzi comment -
Kaosium, you understand what samples were run in the lab on December 18. December 18 is also the day that Stefanoni and some of her lab staff went back to the cottage in Perugia to recover the bra clasp. Perugia is at least two hours driving time from Rome, where Stefanoni's lab is located.

We should know from the time stamp on the bra clasp collection video what time of day Stefanoni was videorecorded in Kercher's room in Perugia. Can that time be compared to any known times when items were tested in her lab in Rome? Did Stefanoni claim that she did the testing herself? If there is a conflict with the times, can Stefanoni be in two places a hundred miles apart at the same time?

 

[Strozzi]

Or you could just go here:

http://murderofmeredithkercher.com/batch-two-kitchen-knife-36b-suppression-90-profiles/

Also, see the end of this: http://murderofmeredithkercher.com/...en-samples-early-exclusion-sollecito-lumumba/

I would like to ask those who have looked at the lab data of evidence such as blood samples collected outside the downstairs apartment and inside it the following:

1. Do you believe a human being dripping blood entered the downstairs apartment after the murder?
2. Do you believe the person is male or female?
3. Do you believe that some of the blood drop patterns indicate the person may have been wearing clothes wet with water!
4. Do you believe the person lay down on a bed?
5. Do you believe the person used the downstairs shower?
6. Do you believe Stefanoni understood it was a human being and not solely a cat?
7. Do you believe Stefanoni suppressed evidence of this?

 

[Kaosium]

This is what I believe is at the heart of the lab work deception. Stefanoni doesn't want conclusive proof...she wants a "shadow of doubt" to appear to be possible. In fact Mignini bases his whole case on exactly this type of deception.

The Qubit is tossed in so as to create confusion and avoid the EDF record trap she would be in if she just processed it all on the one machine.

It is notable that Stefanoni was unqualified to do LTN/LCN work. Her lab was neither certified or equipped to do LTN/LCN work. And especially in 2007 the scientific community voiced opinion against using such suspicious and small data samples in criminal cases at all. (They still do not recommend it)

The main effect of using the Qubit and avoiding Real Time was that she'd never have a quantification figure. This allowed her to claim before Micheli that the amount was some 'hundreds of picograms' which, while smaller than a typical sample, would have been above the low template threshold whether you figure that at 100 pg or 200 pg. As a result no one can say with any certainty just how big the knife blade sample is, just that according to the NIST data her results for the knife blade (bottom one) correspond best with the data from the ten pg samples using a 28 cycle Identifiler kit which was what she said she used in Massei.

This might make you even more alarmed by Low Template DNA being used, but believe it or not this isn't the worst application of it I've ever seen. A year or so ago I came across the McCann case and it just so happened Portuguese officials managed to top Stefanoni for misuse of low template DNA analysis. They found what they claimed was a 'partial profile' of the poor little girl from a mixed sample with at least three contributors where a little more than half of them were consistent with her profile. In a trunk her parents accessed and a sibling may also have been, not to mention clothes and stuff from her could have been kept.

So they called it a 'partial profile' of the little girl and 'evidence' the parents stuffed her in the trunk.

 

[Kaosium]

Kaohsiung comment
. . . I looked it up, those other samples weren't run until December 18th, 2007:

Originally Posted by C&V
It should be emphasized that from an examination of the Real Time PCR reports exhibited, it appears that quantification using this method was only carried out, on 18 December 2007, on the DNA extracts indicated with the following numbers:

48649 = sample D

48651 = sample E

48654 = sample F

48655 = sample G
__________________
"Honi soit qui mal y pense."


Strozzi comment -
Kaosium, you understand what samples were run in the lab on December 18. December 18 is also the day that Stefanoni and some of her lab staff went back to the cottage in Perugia to recover the bra clasp. Perugia is at least two hours driving time from Rome, where Stefanoni's lab is located.

We should know from the time stamp on the bra clasp collection video what time of day Stefanoni was videorecorded in Kercher's room in Perugia. Can that time be compared to any known times when items were tested in her lab in Rome? Did Stefanoni claim that she did the testing herself? If there is a conflict with the times, can Stefanoni be in two places a hundred miles apart at the same time?

Oh, I believe she ran those results then, that's not the issue and it hardly surprises me she finally became interested in the rest of the samples at the same time they decided to try to gather more evidence.

What I do find surprising is she wasn't interested in those samples before that! Here she had a number of samples that returned the same result as 'B' which she claimed was evidence from the murder on the knife, but had no interest in the rest of the samples which you'd think would have just as good if not better chance of retaining DNA.

Unless of course she knew they wouldn't show any DNA because she knew she'd just profiled some lab contamination and the knife was just a 'smokescreen' or 'BS' as both teams of defense lawyers (who were independent of each other at this point) told their clients as reflected in Raffaele's diary and the 'I was there' prison conversation between Amanda and her parents which was tapped by prison officials.

In other words she never expected initially that knife would see court, it was only when they didn't get the confession they were expecting and needed it that they tried to pretend they did a legitimate forensic analysis.

 

[Kaosium]

Thank you for the info. So the Qubit DOESN'T amplify the sample at all? It only tells us if there is any DNA to begin with?

Exactly, it's a handheld device actually. It looks a little like something from Star Trek.

 

[MichaelB]

Gott im Himmel!

I assume the page is not finished as I cannot find a link to that video thus I'm guessing it hasn't been linked yet. Or I'm just blind!

This is just staggering. To think that Maresca participated in this travesty, running interference as Mignini tried to take the lives of two innocents to preserve the fantasy Mignini created which minimized the role of the man who raped and murdered the daughter of his clients.

The cat blood is interesting, I was not aware that there were so many traces that registered human DNA, my initial thought was perhaps human DNA had gotten into cat blood, but the totality of the evidence suggests that's unlikely. Has anyone put together a map of where this all is, as in where the DNA and blood stains were found? I've a mental map of the cottage and where everything fits there, but it's a void on the outside except for the window of course.

Here's to you and everyone involved responsible for this!


I love that scene, nothing quite like solving a problem with the proper application of a gigajoule or two!

This page has the photos.

http://murderofmeredithkercher.com/blood-evidence-downstairs-apartment/

 

[Kaosium]

This page has the photos.

http://murderofmeredithkercher.com/blood-evidence-downstairs-apartment/

Thank you, Michael.

Regarding that interior door seen around 6:36, did police smash that in? Did they determine that it was already like that and unrelated to the murder?

They blamed that mess on a cat and blood from insects?

 

[acbytesla]

Exactly, it's a handheld device actually. It looks a little like something from Star Trek.

I've seen pictures of it. I'm getting a better idea how this all works. This reminds me of when I just started working in the data communications industry when every word and acronym and process was new to me. Back then. I remember thinking I'd never understand it. Now, that is all easy for me. And when a new product comes out in the industry I learn it in minutes...back then it was like oh no... how does this new thing work?

Learning the DNA right now seems exactly the same. It's like learning a new language and you have to learn enough of the basics to make learning the rest relatively easy.


To me, the problem with learning all of this is understanding the language and terminology. All the descriptions sort of assumes that the reader understands the process. That we know what PCR, quantitation, quantification,amplification and what the dyes are and what it means to bind to double DNA and what eletropherisis is and what gene sequencing is and what STR is and what is a haplotype is and what the Speed vac is or the Qubit or the Real Time etc etc. I really wish someone with all this test equipment made a You Tube video and went through the process step by step describing each machine and each step in plain English and then used the nomenclature.

I finally feel like I'm over the hump with it although there still is some blanks to fill in. For example, why does Stefanoni use the speedvac to concentrate the sample? (The speedvac seems to use centrifuge and heat to dry up the liquid in the well while not destroying the DNA...right?) Is this really necessary?

And why is the quantification so important? In other words, I have a good profile, why is it so important to know precisely how much DNA is in the sample? That seems superfluous.