Continuation Part 11: Amanda Knox/Raffaele Sollecito

[Chris_Halkides]

Following up luminol or fluorescein with colorimetric tests

I decided to list out the various lines of evidence indicating that a colorimetric test usually follows a luminescent test. I’ll comment more as time permits.

One, Mrs. Stefanoni performed a TMB test after the luminol test, and her testimony indicated that a negative TMB was grounds for concluding that a the substance was not blood. Two, Sarah Gino indicated that TMB was positive in about half of the luminol-positive areas in her experience. Three, the chapter by Gefrides that a couple of us have cited indicated that this is the norm. Four, even a paper that the PG commenters usually likes to cite (Castello et al., Canadian Society of Forensic Scientists, 2002, 35(3), 113-121) notes that a second test is usually recommended, even though these authors seem to disagree with this recommendation.

Five, A technical bulletin on luminol notes that, "Luminol is only a presumptive test and should be used in conjunction with a second field presumptive test and followed by laboratory analysis if sufficient amounts of staining are detected." Six, Anders Nilsson wrote, “At the present more or less all used forensic blood tests has a limited selectivity and are presumptive. Due to the lower selectivity of the luminol test positive reactions should be verified by other presumptive blood tests like the phenolphthalein test [14].” He also wrote, “The luminol test’s effect on the subsequent use of presumptive blood tests based on colour changing compounds has been investigated at several occasions.” These investigations would not have been performed if people did not follow luminol with other tests.

Seven, “After any positive reaction with luminol, the stained area should be circled with a grease pencil or other suitable marker. The stained area should be checked again with another reagent for blood such as tetramethybenzidine (TMB), phenolphthalein, or o-tolidine. The luminol preparations do not induce a false positive reaction with either TMB, o-tolidine, or phenolphthalein (Laux, 1991).” This passage is on p. 162 of Interpretation of Bloodstain Evidence at Crime Scenes. 2nd ed., (1998) William G. Eckert and Stuart H James, editors.

 

[Kauffer]

And why then, Hemastix is among the "most commonly used by forensics", while other versions of TMB are not?
Did you consider that perhaps Stefanoni would run a Hemastix test precisely because it is "the most common" among forensics?
And maybe, this is so common also because of the so frequent unjustified scientific habits (that occur on many disciplines) like the diffuse protocols encouraging the "colorimetric" second test as reported in wrong handbooks?

And also, did you realize that LJ provided no scientific literature in support of his claim that the two step test is "hugely more specific" than luminol?

Well, I'm playing catch up a little with some of the science; others here do it for a living. Nevertheless, we can see from cited sources that a fluorescent test should be followed by a colourimetric test. These are presumptive tests. If either is negative there would appear to be no need to proceed to a confirmatory test. It isn't blood. Otherwise you must proceed to confirmation. There's a cost saving issue here as well. Laboratory time is expensive. If you can rule out blood at a crime scene, you don't waste valuable resources later.

Since your citations involve a discussion of Hemastix, I doubt they have any relevance. However there is nothing you have shown that contradicts as a relevant procedure the combination of luminol and TMB testing at crime scenes or the absolute requirement to proceed to confirmatory testing in order to confirm the presence of blood.

I imagine that LJ is on the money with regard to specificity. Let us see.....

 

[LondonJohn]

And why then, Hemastix is among the "most commonly used by forensics", while other versions of TMB are not?
Did you consider that perhaps Stefanoni would run a Hemastix test precisely because it is "the most common" among forensics?
And maybe, this is so common also because of the so frequent unjustified scientific habits (that occur on many disciplines) like the diffuse protocols encouraging the "colorimetric" second test as reported in wrong handbooks?

And also, did you realize that LJ provided no scientific literature in support of his claim that the two step test is "hugely more specific" than luminol?

This is ridiculous. The majority of false positives with Hemastix come from plant-based peroxidases, the chlorites in bleach, permanganates and certain metal salts, all of which are capable of catalysing the oxidation of the TMB chemical (thus causing the colour change) on their own. On the other hand, the haemaglobin in blood needs the presence of peroxide (usually hydrogen peroxide) in order to cause the heme ion to cleave the O2 from the peroxide and thus catalyse the TMB oxidation.

Therefore if you can separate the two processes (the application of TMB and the application of peroxide), you can tell whether the TMB colour change is being caused by the test substance alone or whether it's being caused by the test substance only in the additional presence of peroxide. If the former, then the test substance is one of the Hemastix false positives (peroxidases, bleach etc), but if the latter, it's presumptively blood (but still requires a mandatory confirmatory test).

Ergo: the two-part TMB test is by definition more specific than Hemastix.

 

[LondonJohn]

Well, I'm playing catch up a little with some of the science; others here do it for a living. Nevertheless, we can see from cited sources that a fluorescent test should be followed by a columetric test. These are presumptive tests. If either is negative there would appear to be no need to proceed to a confirmatory test. It isn't blood. Otherwise you must proceed to confirmation. There's a cost saving issue here as well. Laboratory time is expensive. If you can rule out blood at a crime scene, you don't waste valuable resources later.

Since your citations involve a discussion of Hemastix, I doubt they have any relevance. However there is nothing you have shown that contradicts as a relevant procedure the combination of luminol and TMB testing at crime scenes or the absolute requirement to proceed to confirmatory testing in order to confirm the presence of blood.

I imagine that LJ is on the money with regard to specificity. Let us see.....

I am right on the money

And of course you also illustrate the most important point, which in fact renders the arguments about presumptive tests somewhat moot: you can't state that the test substance is blood unless and until you have a positive confirmatory test. All of the presumptive tests are just points on the roadmap towards a confirmatory test, with the idea being that the expensive confirmatory tests are only conducted if the escalating series of cheaper and easier presumptive tests have shown positives first.

Therefore just as it's somewhat irrelevant whether the Perugia goons' post-Luminol-positive TMB test was Hemastix or 2-part TMB (if it was Hemastix then they are grossly incompetent, and if it was 2-part TMB then they may have mendaciously concealed false negatives), it's critically important that they presented no positive confirmatory tests for blood to the courts (or the defence......). As Prof Boettcher stated in the expert letter I quoted from earlier, "a positive result for blood in a presumptive test cannot, and should not, be claimed to identify the presence of blood." Simply put, you need a confirmatory test positive, otherwise you can't claim to have blood.

 

[Kevin_Lowe]

In fact, at page 14 the report says:

It says, the 21:10 interaction is likely just the moving of the Amelie film into the "viewed" folder, as a consequence of the fact that the movie was at the end.
While Amelie and many Stardust version were viewed (presumably checked during download) through VLC, there is no "Naruto" file in the "recently viewed" log of VLC; so this one was not viewed with VLC. This is an element that tends to rule out that someone watched Naruto that evening (and, btw, would you watch a "Naruto" episode with your girlfriend?).

The logic is flawed. Suppose we agree that the Naruto file was not played with VLC. How do you get from this to:

Some program accessed the Naruto file, but it was not a video viewer.

There are video viewers other than VLC. For example Quicktime, included on all Apple computers, can play some avi format files with the right plugins. The Naruto episode in question was in avi format.

If you do not think that Raffaele played a Naruto avi with a video viewer, what on Earth do you think he was doing with it? There are indeed other things one might to with a video file, but in the overwhelming majority of cases what one does with a video is watch it.

 

[Samson]

Thanks more or less what I meant.

Therefore the issue is two bare feet need to hit one of Rudy's prints, less chance, rather than one foot, strong chance.
Actually I am surprised this is not discussed more, but it is obviously better not to concede to any blood for the defence, despite this alternative explanation seeming pretty good to me.

 

[Machiavelli]

Well, I'm playing catch up a little with some of the science; others here do it for a living. Nevertheless, we can see from cited sources that a fluorescent test should be followed by a columetric test.

And you have also seen from cited literature that the reasons brougnt for this are disproven (since the cited literature does not only deal with luminol and TMB, but with all the main colorimetric tests).
The cited studies conclusions imply that the handbooks are wrong (since contrarily from what handbooks assume, luminol is the best and most important presumptive test under all main aspects).

Since your citations involve a discussion of Hemastix, I doubt they have any relevance.

The studies also point out that it is Hemastix the TMB test that is the most common among forensics, not other TMB tests.
Stop just for a moment to take a breath and think about the implications of this. Stefanoni would be an exception if she didn't run Hemastix. This one is the routine test, why should we assume an exception?
And why then, while Stefanoni would perform a two-step TMB test, all the other forensics would run Hemastix instead, if they thought other TMB tests performed better and were more useful?
How can this notion of Hemastix being the TMB widely used test be irrelevant?

But then, after all, why should LJ's statement about highly superior specificity of other TMB methods receive credit?
The only difference consists in mixing peroxide to the swab in a sequence, peroxide is the oxidant agent in the reaction, so that if TMB (the reductant) reacts with the sample without peroxide, it means there is an oxidant already present in the stain that interfers (it may be catalyzed by traces of a possible another substance, or may not). This is the only information you could infer. Bleach is an oxidant like Peroxide, so this is why, if there is bleach in the stain, you might see the reaction caused by it immediately in the first step so you would detect the presence of an oxidant interfering agent. This is not different from luminol actually, where luminescence reactions from bleach usually can also be distinguished by the naked eye on timing basis, since they are too quick and intense and last for a too short time.

Apart from thisdifferent ways of detecting bleach, there is no evidence of greater selectivity of two-step TMB test over luminol. On the contrary, literature does not report this alleged specificity anywhere.
So where is the basis for LJ's claim?

However there is nothing you have shown that contradicts as a relevant procedure the combination of luminol and TMB testing at crime scenes or the absolute requirement to proceed to confirmatory testing in order to confirm the presence of blood.

In fact,
1) no evidence was brought about such alleged relevance of a combined sequence of luminol followed by TMB test. Such alleged relevance is unsupported and quite made up, obviously disproven by literature, bearing in mind that literature considers Hemastix the most sensitive and common method (but Quickenden and Creamer apparently consider the two-step TMB test as a term of comparison with luminol instead). No trace of the suggestion to perform a second colorimetric test shows up also in the Barni et al. (2006) study on luminol, where "operational use of luminol" is also discussed.

2) no evidence exists that a "highly selective" TMB test was ever performed on the luminol stains.

3) no plausible substance that reacts with luminol but not with TMB is know.

4) no plausible alternative dynamic that explains how the footprints were made was ever presented by anyone.

5) no evidence that a confirmatory test was performed of a kind that would actually rule out something, was ever presented.

6) Your insistence on a concept of "absolute requirement" is a wrong setting for a reasoning about evidence assessment. There is no place for this undue condition within judiciary principles. You may not require pieces of evidence to be probative or to be complete. The judges will consider as the important logical framework only the plausibility of alternative, which is requirement in a hierarchical position of priority that will determine whether a finding is a piece of evidence, before assessing whether it is probative or complete.
The plausibility of an alternative explanation is more important, in the probative reasoning, than those requirements for certainty that are internal to scientific disciplines.

The only contrary argument among those recently presented that has rational structure, is the argument by Plaingale (which appears to be an argument that does not defy reality but instead employs deduction from assuming an "intention" of the prosecution).

 

[Machiavelli]

There are video viewers other than VLC. For example Quicktime, included on all Apple computers, can play some avi format files with the right plugins. The Naruto episode in question was in avi format.

However, there is no Quicktime log that supports the defence alibi. And we may also note how all other videos appear to be viewed with VLC.

If you do not think that Raffaele played a Naruto avi with a video viewer, what on Earth do you think he was doing with it? There are indeed other things one might to with a video file, but in the overwhelming majority of cases what one does with a video is watch it

.

Well, frankly, I don't know if he was doing things with it. How can you access a file through other applications? Well, I am not a computer expert, but besides playing a video, it seems an application may move a file from a folder to another, restore or conclude a download.

 

[Machiavelli]

This is ridiculous. The majority of false positives with Hemastix come from plant-based peroxidases, the chlorites in bleach, permanganates and certain metal salts, all of which are capable of catalysing the oxidation of the TMB chemical (thus causing the colour change) on their own. On the other hand, the haemaglobin in blood needs the presence of peroxide (usually hydrogen peroxide) in order to cause the heme ion to cleave the O2 from the peroxide and thus catalyse the TMB oxidation. (...)

No scientific papers reports nything of the kind. You are making your own scientific speculations and guesses, and they are wrong. The oxidation of TMB chemical cannot take place (be catalyzed) in the absence of an oxidant, and the oxidant is precisely the peroxide that you add on the second step. What reaction can the vegetable peroxidases and metal salts catalyze, until there isn't the second reagent?
Metal salts do catalyze the luminol reaction as well as the TMB reaction because their ions can be used, again and again, but you need to have two reagents, not just one. You must have an oxidant. TMB does not change colour without an oxidant. Catalyzers are not enough.

 

[Chris_Halkides]

the scroll tool is almost as good a friend as Google is

3) no plausible substance that reacts with luminol but not with TMB is know.

This statement is suggestive that you are not bothering to read what others have been writing. Scroll up and you will find some.

 

[Machiavelli]

Indeed: I say exactly the same thing on this point, that luminol was found by multiple studies to be far more sensitive and nor specific than TMB and having the same false positives, "as if it hadn't been refuted", I say this precisely because it was not refuted. And it can't be refuted. You can't refute, as a matter of facts, that authors like Webb et al. and Tobe et al. made these findings.
The fact thar Quickenden, Creamer et al. pr others set higher or narrower theoretical limits, doesn't change a iota about the validity of those researches.

The content of those alleged cleaners is bleach. It is implausible not only because no instance or trace of the presence of those products was found at the apartment, but also because it is implausible that people walk barefoot in bleach; and even if they did so, it would be unexplained why there are only isolated bare footprints, not in a trail, instead of for example a messy set made of many trails, maybe in negative prints on a cleaned luminescent floor, and why only in that area of the house; and then, that would also leave the bloody prints unexplained or commend their explanation to an inconsistent scenario. Another element too, is that luminescent reaction from oxidants like bleaches are distinguishable from blood since their luminescence lasts for a much shorter time.

As for the soils: first things to say, also true for bleaches, is that metal salts dilutions in soils are reactive to TMB as well, something that anyway would demolish the pro- Knox TMB argument.
Second thing, it would be extremely easy for the defence to prove that soils around the cottage have this property. They didn't bring in this simple evidence.
Third, if it's something so common as soil, instances of it would be found about everywhere in the house, the more external areas in particular, there is instead a very limited number of instances, only in a certain area of the house but with a so peculiar distribution.
Fourth, if it was soil containing metal salts, you would find a lot of shoe tracks, coming from the outside, instead of naked footprints (which normally come from bathrooms). If the tap water contained reactive iron you would find thus evidence about everywhere, and you would also see the traces of rusty water with naked eyes since iron content used repeatedly tends to form visible traces.
Fifth, it dies not explain why these metal salts have the shape of one if Guede's shoeprint (the missing part of it, near Meredith's door).
Sixth, if doesn't explain as a probable finding the presence of mixed DNA of victim+suspect where none of them was supposed to be found, and dies not respond to the analogy with the bloody bathmat print.

So none of the proposed alternatives is plausible or strong compared to the incriminating indication.

This statement is suggestive that you are not bothering to read what others have been writing. Scroll up and you will find some.

You always talk about bleaches. Maybe you didn't bother to read what I wrote yesterday, again. It is not true that bleaches are reported as non reacting with TMB, and anyway bleaches are not plausible.

 

[MDDVS]

You always talk about bleaches. Maybe you didn't bother to read what I wrote yesterday, again. It is not true that bleaches are reported as non reacting with TMB, and anyway bleaches are not plausible.

Stop with this nonsense, there is nothing incriminating about the luminol hits even if they were blood.

 

[js202]

First things, first

Stop with this nonsense, there is nothing incriminating about the luminol hits even if they were blood.

Indeed, as Kauffer has authoritatively argued on epistemological grounds, unless and until PG types - even self-proclaimed nemeses - can prove a clean-up in the murder room itself, which left Guede's traces and removed all traces of AK and RS, these luminol blobs are bootless nonsense. And John Douglas, amongst other experts, has authoritatively argued that the blood smears coupled with the size of the room leave no space for second or third participants, much less a clean-up.

I mean, at the end of the day, who are you going to believe: an obsessive-compulsive, anonymous loon on the internet, or a storied pro with first hand knowledge of thousands of murder scenes?

 

[Desert Fox]

You always talk about bleaches. Maybe you didn't bother to read what I wrote yesterday, again. It is not true that bleaches are reported as non reacting with TMB, and anyway bleaches are not plausible.

The soil in Perugia can trigger luminol
It contains umber

 

[Chris_Halkides]

the lack of DNA is a problem for the prosecution

Thanks more or less what I meant.

Planigale and Samson,

I don't wish to say with 100% certainty that they are not blood at this time, but here are a few of my reasons for thinking that they probably are not blood from the crime. One, Meredith's DNA is not present in the majority of the spots, yet low template DNA profiles can be generated from only a few cells. Two, the prints in Amanda's room do not match her reference foot. Three, the print in the hallway assigned by the FP to Raffaele does not look like his foot (see MichaelB's comment a photo a few days ago).

I think that there is another more plausible hypothesis, namely that a substance has become bonded to the floor is causing the luminol reaction. Luminol penetrates through paint, according to a citation given earlier this week. The lack of reactivity with TMB might be because the overapplication of luminol created a situation where the test for TMB failed due to a failure to acheive the correct pH. Luminol is done under basic conditions, but the TMB test is done under acidic conditions. I am also working on another possibility.

 

[Bill Williams]

Indeed, as Kauffer has authoritatively argued on epistemological grounds, unless and until PG types - even self-proclaimed nemeses - can prove a clean-up in the murder room itself, which left Guede's traces and removed all traces of AK and RS, these luminol blobs are bootless nonsense. And John Douglas, amongst other experts, has authoritatively argued that the blood smears coupled with the size of the room leave no space for second or third participants, much less a clean-up.

I mean, at the end of the day, who are you going to believe: an obsessive-compulsive, anonymous loon on the internet, or a storied pro with first hand knowledge of thousands of murder scenes?

It is not just that the nemesis has it as his method of proof, the troubling thing is that both Cassation and the Florence court headed by Nencini also seems to have it....

..... which goes like this:

The prosecution does not need to prove that there is was a clean-up in Meredith's room, to explain why Rudy Guede's forensics were found there (and in the victim) and that AK and RS's were not:

The absence of those sets of forensics must bee seen in the light of the whole crime scene which includes the whole upper floor of the cottage.

Therefore, given that the whole crime scene does not rule out AK or RS, then is is logical to assume that the absence of AK' and RS's forensics in Meredith's room must have some explanation, therefore they are guilty.

(I think I am getting the hang of this.....)

I am almost positive that Italian law in general does not work this way, and certainly not in the vast majority of cases. But for some reason, they've decided to judge things this way for this case.

 

[moije2]

Is there actually a "Machiavelli Room" in the basement of the Perugia Questura, filled with computers and fully staffed with beefy, mustachioed Italian researchers pounding away at google screens to gather and analyze all the info our Mach seems to have at his fingertips?

Is anyone else amazed at this display of renaissancian dexterity?

Well I'll say it if no one else will; Leonardo, Michaelangelo, and Machiavelli!

I am partially at least enjoying this display of committee activity, and by that I mean the Ministry of Machiavelli. I imagine by this flurry of activity that there must be some sort of holiday where they are located that has allowed them to convene and organize their responses. Yes I am amazed. A bit.

The question is, why?

They are putting enormous energy into battling the scientific community in order to...? Defend not-a-real doctor Stefanoni against incompetence? The highly trained and qualified "professional" that destroyed the bra clasp evidence? To somehow prove that the dilution of blood allows for the "presumptive" tests to slip through a narrow window to allow the "blobs" to be blood? When it is irrelevant given the traipsing through apartment?

Yes, perhaps there is a steamy smoke filled basement basement with those types that you suggest!

 

[Dan O.]

In layman terms this would mean a blood dilution of about 1:2million/2.5million, since a red cell would contain 27-30 picograms of Hemoglobin.

But the protocol for the reading of ABAcard requires that a small fraction of the collected swab (no more than 3 square millimeters) is placed into a 300 microliter buffer solution and micro-centrifuged, and and it's this final solution what will be tested and needs to have the 0.05 ug/ml hemoglobin concentration.
So a sample would undergo this proces of reduction and dilution before being tested.
It is anyway unknown whether the ABAcard test was used, or whether a confirmatory test was performed at all.

If you don't know what the big words are you should look them up before posting.

 

[Kevin_Lowe]

However, there is no Quicktime log that supports the defence alibi.

Nor is there any substantiated lack of such a log. You seem to think that it's up to the defence to prove their case beyond reasonable doubt. It's the other way around, the prosecution has to discredit the defence beyond reasonable doubt. If the computer log says that Raffaele was at home playing a Naturo video at 21:10 surely it's up to the prosecution to prove that wrong, not up to the defence to prove beyond all possible doubt that it's right?

And we may also note how all other videos appear to be viewed with VLC.

So? Some video formats can be played on VLC but not Quicktime. Is downloading different videos from different internet sources in different formats is evidence that someone is a murderer somehow?

This reminds me of the ludicrous contortions pro-guilt posters went through in the early days to convince themselves that it was absurd that a person would scoot on a bathmat to avoid dripping on the floor. Some people would see it as evidence of murder if Knox or Sollecito put their socks on before their shoes.

Well, frankly, I don't know if he was doing things with it. How can you access a file through other applications? Well, I am not a computer expert, but besides playing a video, it seems an application may move a file from a folder to another, restore or conclude a download.

So you admit that he could perfectly well have played it with a different application, and that this is a far more likely thing for a person to do with a video file than anything else?

 

[Dan O.]

In fact, at page 14 the report says:



It says, the 21:10 interaction is likely just the moving of the Amelie film into the "viewed" folder, as a consequence of the fact that the movie was at the end.

I didn't know anyone was arguing that the last access to Amelie wasn't an automatic response to the movie ending. But changing the location of a file would change it's inode and therefore cause a cooresponding change to the files last change date. What was the last change date of Amelie?

While Amelie and many Stardust version were viewed (presumably checked during download) through VLC, there is no "Naruto" file in the "recently viewed" log of VLC; so this one was not viewed with VLC.

Many applications keep a recent files list and I've notices a particular anomaly that the list typically only includes those files that were opened by the user from within the application. When the file is opened by clicking on it's icon within the Finder, the application's list does not get updated. Has this behavior of VLC been verified?

Another possibility is that the video was viewed from a different application. I believe it was noted that Front Row was active. For many of the videos I view following this case I prefer to open them in QuickTime even if they are using the CODEC from VLC. The VLC application is quite buggy.

This is an element that tends to rule out that someone watched Naruto that evening (and, btw, would you watch a "Naruto" episode with your girlfriend?). Some program accessed the Naruto file, but it was not a video viewer.

And you too aparently don't know the difference between the Last Opened date maintained by Spotlight and the Last Access date maintained by the file system.

The "windowserver" argument on the other hand is even worse evidence, since an open VLC window (even without anything being viewed) with default setting would have kept the keyboard active and prevented the screensaver from running.

I pointed that out as a possibility way back but not having that version of VLC to test I could not verify it. If you look at what the experts were doing with similar questions, they didn't speculate on how the preferences were set. They looked at the actual settings and checked the timestamps of the settings file itself to insure that it had not been altered since the night. Either those experts are realy smart or they were following this thread and read my post on the subject.