Tuesday, March 16, 2010

Luminol and some of the footprint evidence

Part X in a series on the Knox/Sollecito case

The story surrounding the footprints in the murder of Meredith Kercher is complex, and it may take more than one post to cover it. However, the recent jury’s statement included a reference to the footprint evidence as one indicator of guilt. In this post we will summarize the analyses of both pro-prosecution and pro-defense points of view with respect to a bloody footprint found on a bathmat and the three luminol-positive footprints in the hallway.

Chemistry and forensics of luminol
Luminol was used to visualize some of the footprints. Luminol is typically used as a mixture that includes sodium carbonate and sodium perborate. Luminol reacts with the iron atom in hemoglobin, and also reacts with other substances, such as bleach and fruit juice, or with substances that contain a metal ion that can catalyze the light-producing chemistry. Fruit pulp is rich in certain peroxidase enzymes that have an iron ion that has much in common with the iron in hemoglobin. In hemoglobin and in the peroxidase family of enzymes the iron ion is bound to four nitrogen atoms of an organic molecule called protoporphyrin IX. The combination of iron and protoporphyrin IX is called heme, which is tightly bound to the protein portions of hemoglobin or catalase, respectively. A fifth nitrogen atom from a histidine residue within the protein also coordinates the iron ion (Frey and Hegedus, Enzymatic Reaction Mechanisms, pp. 203-209). Heme is called a prosthetic group, a nonprotein molecule that helps a protein to do its job.

Both hemoglobin and peroxidases are proteins, but only peroxidases are enzymes (biological catalysts). In the luminol reaction however, both hemoglobin and peroxidases are acting catalytically. A paper by Barni et al., “Forensic application of the luminol reaction as a presumptive test for latent blood detection,” Talanta 72 (2007) 896–913, is a review of luminol chemistry and forensics. Hydrogen peroxide oxidizes luminol with the production of light. The iron ion within hemoglobin or other substances is a catalyst; in other words, one molecule of hemoglobin converts many molecules of luminol and hydrogen peroxide to produce 3-aminophthalate. Thus the catalytic behavior of metal ions partially explains the sensitivity of this test. Since photons of light are emitted in this reaction, the process is classified as a chemiluminescent reaction.

This paper discusses whether one can tell whether or not the substance reacting with luminol is really blood:

“Due to the possible presence of these substances at the crime scene, the luminol test must not be considered sufficiently specific to permit an unequivocal identification of blood [15,18,51,88,89]….

Generally visual examination is used when the luminol test is employed in a forensic situation, rather than instrumental detection of the luminescence. An experienced practitioner may distinguish the true blood-catalyzed chemiluminescence from that produced by other substances by the evaluation of parameters observable to the naked eye such as emission intensity, duration and spatial distribution. However this approach may also lead to misinterpretation, due to a subjective, informal and non-quantitative evaluation, for example, because its intensity is qualitatively much weaker than that expected for blood. In other circumstances an emission of similar intensity may be thought to derive from diluted bloodstains and is accepted. Therefore, caution should be exercised when using the test.”

Thus the Luminol test is a presumptive test and should be confirmed by one that is more specific for blood. However (http://www.friendsofamanda.org/luminol.html), “The prosecution's key forensic witness told the court she can tell by looking at a luminol reaction whether it involves blood or something else, but she did not perform any scientific tests to validate this claim.”

The prosecution attributed one hallway footprint to Raffaele Sollecito. Francesco Vinci testified that the luminol footprint in the hallway is a human footprint but one that cannot be attributed to anyone in particular (http://perugia-shock.blogspot.com/2009/09/sollecitos-expert-questions-all.html). The shape of the big toe and the print of the putative second toe were key elements to his conclusions. Frank Sfarzo wrote, “With such limited elements Vinci is only able to rule out Raffaele (or the girls) and to say that the print is compatible with Rudi. And we can only admire his honesty and consistency.”

Additional posts on the footprints by Frank Sfarzo

Darkness Descending
Now let us turn to the analysis of the footprints that Colonel Luciano Garofano gave to the author of the book Darkness Descending, by Paul Russell and Graham Johnson (Pocket Books, 2009). Colonel Garofano is a well-known forensic scientist who recently retired from the Carabinieri. He was much friendlier to the prosecution’s case than to the defense’s case, and I do not agree with everything that he said in this book, particularly with respect to the footprint on the blue bathmat discussed below.

“The other problem I have is the way the Luminol was applied. The size of the blobs shows that it was not carefully vaporized but squirted. That creates two problems. It dilutes the sample and it dilates the print. We have a print attributed to Sollecito, which matches his foot in the size of the big toe, the width of the metatarsus and the width of the heel, but does not present the characteristic details each of our feet present. The print can be said to be compatible, but not 100 per cent.”

“Now let’s have a look at the prints attributed to Amanda Knox. There’s one in her bedroom facing the exit to the room, and there are two right feet in the corridor walking in the direction of the victim’s room. The same goes here. The method of application of the Luminol is insufficiently subtle to positively identify a foot, but the result can be said to be generally compatible with Amanda Knox’s.

“But I didn’t see who else they compared the prints with. Just Rudy, Amanda, and Raffaele? So we only have a choice between those? We don’t have the footprint of other women or men, as a comparison? Pity.”

Colonel Garofano believed that it is likely that Raffale’s foot made the print on the blue bathmat, but he did not sound certain. However, he did not discuss the fatal criticism that the prosecution’s expert witness made a serious error in the measurement of this print, discussed below. Colonel Garofano did not address the fact that Rudy’s big toe looks nothing like Raffaele’s, and the bathmat print. Nor did he seriously entertain the possibility that the prints are not even blood. He also did not explain why the print attributed to Ms. Knox points toward Meredith’s room and why there is not a full set of prints. These are serious omissions in that it is difficult to picture how the prints could have been made during the commission of the murder.

Science Spheres
Mark Waterbury discussed some of the problems in the footprint evidence in the hallway and elsewhere (http://www.sciencespheres.com/2009/10/methods-of-polizia-pseudoscientificaa.html):
“Luminol glowing footprints were found in a hallway, and some may have been Amanda's, it is hard to know for sure because they were only compared with her feet, and found to be ‘compatible.’ Again, no controls. Meredith, Laura, Filomena, none of the other resident's feet were compared to these footprints. The footprints were tested for blood, and it came out negative. No blood. So, why are they important? Amanda lived there, after all.”

“Amanda's DNA was said to be found in one of these footprints. Did they also test a meter away from the footprints, to see if her DNA was all over the apartment where she lived? No. That would have been another control experiment. Was the DNA actually associated with the footprint, or did it just happen to be there, because the resident's DNA was all over their apartment, as people's DNA usually is? We will never know. They skipped the control experiments, and presented results without any reference.”

With respect to Meredith’s DNA and the luminol-enhanced footprints, I would argue that not finding her DNA is evidence against the proposition that the substance is her blood. However, finding her DNA would be not conclusive evidence that the substance is her blood. That is because there could be non-blood derived biological material from Meredith mixed into the footprint. In other words finding her DNA would have been consistent with blood being responsible for the luminol reaction but would not demand that conclusion.

Perugia Murder File
A powerpoint, Dear-Mr-Marriott-I-Shrunk-the-Black-Kid.pps, authored by Kermit (http://www.perugiamurderfile.org/viewtopic.php?f=5&t=36) attempted to rebut arguments presented at the Friends of Amanda website. The first of these is that the prosecution’s expert witness Mr. Rinaldi, made a serious error in his size measurements. Kermit’s presentation also addresses two of the problems in ascribing the bloody footprint to Mr. Sollecito, the narrowness of the big toe (http://www.friendsofamanda.org/footprint.html) and the appearance of a mark near the big toe. Mr. Guede’s big toe is shorter than his second toe, but Mr. Sollecito’s second toe does not even show up in his footprint. Mr. Guede’s big toe is narrower than Mr. Sollecito’s. According to Kermit, the reason that the bloody imprint of the big toe seems narrower than Mr. Sollecito’s is that the raised tufts of the blue bathmat have picked up more blood than the unraised portions. Likewise, the mark Sollecito’s expert witness Francesco Vinci would ascribe to a second toe (http://perugia-shock.blogspot.com/2009/09/sollecitos-expert-questions-all.html), Kermit claims is Mr. Sollecito’s big toe. Kermit said elsewhere that his reason for writing his powerpoint presentations were to refute the lone wolf theory of the crime, because the prints were of varying sizes.

I do not find Kermit’s arguments about the raised tufts to be convincing. There is a raised tuft of lighter color in between the big toe and the second mark, possibly from Guede’s second toe. If Kermit were correct, that portion of the bathmat should be as dark as the rest. The validity of Kermit’s measurements has also been questioned by two commenters at a forum at James Randi Educational Foundation (http://forums.randi.org/showthread.php?t=161229&page=126).

Defense-friendly blogs
Charlie Wilkes of the website Friends of Amanda presented the argument that the bathmat footprint measurements made by the prosecution’s witness, Lorenzo Rinaldi, contained a critical error. (http://www.friendsofamanda.org/footprint_measurements.html). Mr. Wilkes also discussed the footprints elsewhere (http://knoxarchive.wordpress.com/2009/11/27/manuela-comodi-asks-for-common-sense/). The site Injustice in Perugia gives a detailed account with images of the footprints (http://www.injusticeinperugia.org/footprints.html). These two sites make several telling points, especially with respect to the luminol-positive footprints in the hallway. The footprint L9 (in the knoxarchive numbering scheme) is attributed to Amanda by Rinaldi. Yet is alone, and it points toward, not away from Meredith’s room. L6 and L7 are two right feet, one of which is attributed to Raffaele. The footprint in the hallway attributed to Raffaele look quite indistinct to me. The other one is attributed to no one at all, and one wonders to whom it belongs. None of the footprints tested positive for blood and none were positive for Meredith’s DNA.

None of the three footprints were part of a trail. It is difficult to see how Amanda Knox and Raffaele Sollecito could have removed other footprints (as part of an alleged cleanup) without also cleaning up Rudy Guede’s nearby bloody shoeprints.

Prosecutorial tunnel vision
The failure to obtain reference footprints is disturbingly reminiscent of the lack of reference DNA from Laura or Filomena, the other two flatmates. It suggests that once the investigators locked onto Amanda and Raffaele, they did not reopen their field of vision. Crime journalist Mario Spezi said about the word “compatible,” in Douglas Preston’s book, The Monster of Florence, which covers a serial murderer in that city.

“Compatible, not compatible, and incompatible are the baroque inventions of Italian experts who don't want to take responsibility. Using 'compatible' is a way to avoid admitting they haven't understood anything. Was the bullet in Pacciani's garden inserted into the monster's pistol? 'It is compatible.' Was that laryngeal break inflicted by someone who intended to kill? 'It is compatible.' Was that painting done by a monstrous psychopath? 'It is compatible.'”

“Perhaps yes, perhaps no--in short, we don't know! If the experts are chosen by the investigators, they say their results are 'compatible' with the theories of the prosecution; if they are chosen by the defendants they say that their results are 'compatible' with the theories of the defense. That adjective should be outlawed!”

Luminol was overapplied to the footprints in the hallway, and a full set of reference footprints was not taken. These two points alone make the prosecution’s attribution of two the footprints to Ms. Knox and Mr. Sollecito, respectively, impossible to accept as solid conclusions. The unattributed footprint forces one to ask who else was in the hallway and when. Moreover, the luminol-visualized footprints did not contain Meredith’s DNA and cannot be said to be blood. The failure to obtain a positive test for blood is possibly the most serious problem for the prosecution’s case. Although I do not claim to be an expert in the analysis of footprints, the bathmat footprint looks like a better match to Rudy Guede than to Raffaele Sollecito. The footprints in the hallway may date from a time prior to the murder, or on the following morning. The lack of reference footprints is one more indication that the forensics in this case was not pursued in an entirely objective manner.


Joe said...


I don't believe any of the luminol-positive footprints had anything to do with the murder of Meredith.

No bare footprints were found in blood in the room, and the prosecution offered no evidence that any footprints were canceled from the room.

There is no continuity of bare footprints from Meredith's room to the hallway and Amanda's room.

The lack of Meredith's DNA in any of the luminol-positive bare footprints and the presence of Amanda's DNA in the three bare luminol-positive prints in Amanda's room are very telling. If these bare footprints were related to the murder, they would have been made with Meredith's blood. If these were cleaned to remove Meredith's blood and DNA, how would Amanda's DNA remain in the three bedroom prints? Would it be humanly possible to remove all traces of Meredith's DNA from all of those prints but leave Amanda's DNA in the bedroom prints? I don't believe it is.

Furthermore, the luminol-positive prints were made on ceramic floor tile. From: http://www.chem.lsu.edu/htdocs/people/rlmccarley/mccarley/Chemistry%202001/Articles_SP2008/Barni_Talanta_Luminol_Forensics.pdf
“Non-absorbent substrate, such as vinyl, tile, glass, metal and many others are fairly easy to completely clean and a mild washing attempt by water and soap lead to the removal of the bloodstains yielding almost non-existent reaction with luminol.” From this scientific article, one can conclude that if a cleanup had occurred as the prosecution alleged, the luminol reactions would not have been as substantial as they were.

One Spook said...

Ok, Chris. My head exploded while reading the first part, Chemistry and forensics of luminol" Back when I took chemistry, we were concerned with trying to turn lead into gold.

I thought the section Defense Friendly Blogs with the link at the "Injustice in Perugia" Blog at http://www.injusticeinperugia.org/footprints.html was excellent and that your conclusions seemed reasoned and sound.

Nice work!

One Spook

Rose said...

Were any of these footprints even tested for blood? I see a lot of statements saying the prints did not test positive for blood which is a bit misleading if they were not even tested.

My initial impression when researching the footprint issue was that none of these prints are Raffaele's and the nature and location of these prints argues against a cleanup (of the floors at least). The lone print in Filomena's room does not make much sense and is an outlier, in my opinion. I still believe the footprints in Amanda's room are related to the moving of the lamp into Meredith's room.

I appreciate you looking into this issue. The footprint evidence is certainly far from conclusive.

Chris Halkides said...


Good point about the tile.

One Spook,

I included the biochemical information on hemoglobin and peroxidase to make more plausible the idea that fruit juice will give a positive luminol result.


I am not 100% sure of the answer to your question. However, if the prints were not tested for blood, it suggests sloppy, biased forensics.


Debrah said...

A riveting post, Chris.

The defense team might need to summon you to Italy.


Randy said...

Not having footprints from the other roommates seems like another example of trying to prove the prosecutor's story and not took place. What do we have that shows these people did it?
Not what do we have and what does it show?

The girls all look to be close in size. Seems like they could have similar sized footprints. It doesn't mean they are not AK footprints but the footprints could also be from the other roommates maybe from a few days before that terrible night.

"When biological samples have to be collected for DNA or other tests, luminol should only be used after samples are seized. Luminol's chemical reactions with blood and other body proteins destroy some important genetic markers required for DNA fingerprinting." from enotes.com

could this be why no DNA was found in the footprints?

Joe said...

could this be why no DNA was found in the footprints?

March 18, 2010 1:11 AM


Amanda's DNA was found in the three luminol-positive prints found in her room.

Chris Halkides said...


A quick perusal of the literature would suggest that luminol does not have a strong adverse effect on DNA forensics. If my opinion changes after more searching, I'll post another comment.


Randy said...

some of the information about DNA and luminol I was reading on google was maybe 10 years old...I thought perhaps not finding Meredith's DNA could have been caused by the luminol possibly changing some of the genetic markers to make it not possible to identify. I found an article earlier today that said now, there are types of luminol that can be used that don't effect the DNA. And I believe something that can be added that cancels the effects of bleach too.

"when the tiles were cleaned with bleach there was an initial drop in chemiluminescence intensity, followed by a rise to a consistently high value, visibly indistinguishable from that of blood. Examination of bleach drying time suggested that any interfering effect becomes negligible after 8 h."

from a study (study to determine the effect on the luminol test when an attempt is made to clean bloodstained tiles with a known interfering catalyst)published in Australia in 2005.

so after some time- 8 hours in this case- the effects of bleach on luminol become negated. But there was not a test result for blood with AK's footprint released. Why is that I wonder? Is this another DNA is there but no blood moment like with the knife? And the prosecutors say "trust us it's there"

Rose said...

From what I have read it seems that the footprints were not tested for blood. That it why I thought it was misleading to say the footprints did not test positive for blood. They were not tested, period. Evidently the investigators felt the Luminol results were enough.

Chris Halkides said...

Results of blood tests from Luminol reactive sites. Luminol glows are only “presumptive” of blood, and are certainly not conclusive. The next step when a glow is seen is to swab the area and use a test that is specific for blood. It was never credible that these simple tests were not done. In fact, they were performed, but were withheld, apparently because they didn't look good for the prosecution. As Sara Gino stated "We were not told that, first of all, the prints were treated with a substance which should have indicated whether they were blood, and the result was very uncertain."

I think I had this quote in an earlier draft of this post, but I cut it out because the post was getting too long. Over at TalkLeft, Seahawk2 criticized this post for the turgid writing, anyway. Such is the sorry lot of bloggers.


Anonymous said...

Chris, That is funny. Good one.

Rose said...

Candace Dempsey has come out of the woodwork to plug her book while at the same time giving us an overtly biased glimpse of the judges report.

You will be pleased that this one is also turgid, bordering on turpid as well as vapid and insipid. I guess yours could be worse Chris.

Turgid Is As Candace Does

Ray Turner said...

Unfortunately Rose, that IS the courts's theory of the crime. It deserves to be ridiculed and it's hard not to be biased. The court's motivation is available to everyone now, which is linked in Candace's article if you didn't notice. I see nothing wrong with pointing just how absurd their scenario is.

Rose said...

Thanks for pointing that out. It is the entire thing, in Italian. Trying to find a program that will rough translate the whole thing rather than 1 page at a time. Good catch. I thought it was a link to her 1st article on the report.

LOL. Can Candace at least make some minor effort to appear objective? Rhetorical, I know, possibly being a bit fervid in my criticism. id going off on a tangent it seems.

Rose said...

Babel Fish looks like it is trying to do it, may take awhile, will let you know. If someone has a pdf transator program perhaps? Or other suggestions?

Ray Turner said...

Thank you for responding. I think we disagree with a lot on this case, but I do enjoy your posts and analysis here and appreciate your biting wit most of the time :). I particularly like this blog's comment section because it has so far been devoid of name calling.

Believe me, there have been several times I wanted to say some very awful things (I am only human) about people from PMF and True Justice, or Barbie Nadeau and Andrea Vogt, but it just feels kind of dirty. Instead, I offered $10,000 to anyone who could find me a post at the TJMK blog that was critical about ANY aspect of the prosecution's case. I am still waiting for someone to try and take this money.

Hey, can we not agree I have the most biased blog though? Just look at the title. : ) At least some people have the honesty to admit their bias.

I just take issue with people who think a writer can't promote their book or editorialize on their own blog. I don't mind if Barbie Nadeau does it. I don't see the big deal. We have straight reporters and those who editorialize.

And let's be honest, at this point, you can name pretty much every writer and say what they really think about the case--except Nick Pisa. I can never tell what he thinks. Lol.--Ray

P.S. Rose, the .pdf won't work in Babel Fish because it won't recognize the words since I believe it's a photocopy. Feel free to email if you want a translatable copy.

Anonymous said...

Hi Rose,

Didn't you call the "Motivations" report "bizarre." A comment with which I entirely agreed?

I don't believe Ms. Dempsey has added or detracted from our basic understanding of the report, has she?

You were correct. The "Motivations" report is bizarre.

And inexplicable, dubious, specious, asanine, doubtful, inapt, unlikely, implausible, preposterous, irrational, unreasonable, wacky, incoherent, fantastical, non-sensical, illogical, far-fetched, ungrounded, disjointed, disordered and nearly manic in its loose and almost laughable associations.

The "Motivations" report would be fabulous had it been produced by a fiction writer like Lewis Carroll. I absolutely would have loved it in that context.

Camus could have done marvelous things with this "Motivations" report.

But as the published (and possibly mistranslated) opinion piece of a judge (a JUDGE!), it bears no resemblance to any legal writing I have ever encountered.

As Gregory used to say, MOO,

Rose said...

Observer said: "And inexplicable, dubious, specious, asanine, doubtful, inapt, unlikely, implausible, preposterous, irrational, unreasonable, wacky, incoherent, fantastical, non-sensical, illogical, far-fetched, ungrounded, disjointed, disordered and nearly manic in its loose and almost laughable associations." LOL. A thing of beauty, Observer.

Ray, it is an image pdf, rats. I emailed you. Maybe Chris could post a link here as well?

Randy said...

here are some interesting comments from an article at foreignpolicy.com

the article was titled "can anyone get a fair trial in Italy? dated 12/10/10.

"At the moment, the Italian public's trust in the justice system is at an all-time low. According to a November poll by Euromedia research group, only 16 percent of Italians fully trust it; just two years ago, the figure was 28 percent..."Inquiries are conducted without any reliable methods," says Roberto Malini, president of EveryOne, a nongovernmental organization that defends ethnic minorities in jail. "Tests take place solely in the laboratories of the state police. There's no independent lab, and independent observers do not have access to the police's work."...Legal experts also share concerns about Italy's bar for admissibility. Il Giornale, a conservative newspaper, for instance, recently published an interview with Marco Morin, a Venice (ahhh Venice!! my favorite city!!!!...ok, that is my comment) -based firearms expert who declared he no longer wanted to work in Italian courts..."Here, they accept everything without questioning, as long as it comes from the institutional laboratory."..."Here in Italy trials take place in TV, rather than in court," Judge Francesco Cananzi, a representative of the national council of magistrates, publicly stated this year."


FOREIGN POLICY is published by the Slate Group, a division of Washingtonpost.Newsweek Interactive, LLC

Anonymous said...

Thanks, Randy. I had missed that FP piece.

Based on what we've seen out of the Italian justice system in this case, I'd say the article is right on point...even if it mistakenly stated that Ms. Knox "confessed" and then retracted the confession. Her statements were never a "confession."

It is nearly incomprehensible why only 16% of the population trusts the Italian justice system, yet in a deeply flawed case the prosecution was able to get a unanimous verdict.

Apparently, though, all were willing to accept wholesale the results of the State Lab, and no one was willing to be on Mr. Mignini's bad side.

I think we can understand why some say the sorry state of the Italian justice system is what led to the development of a shadow "justice" system, namely the Mafia.