Tuesday, July 10, 2012

Presumptive blood testing in the Chamberlain, Taylor, Lovejoy, and Knox/Sollecito cases

Part 34 in the Knox/Sollecito case

Update (9 July 2013)  I renumbered this entry from 35 to 34.

In this post we will examine presumptive testing in the cases against three other individuals before returning to the Knox/Sollecito case.  Presumptive blood testing has been a subject of this series twice before.

Lindy Chamberlain
In 1982 Lindy Chamberlain was tried for the 1980 murder of her daughter Azaria near Ayers Rock in the Northern Territory of Australia.  A presumptive blood test in the Chamberlain’s car was positive:  The ortho-tolidene test is similar to the tetramethylbenzidine (TMB) test in that both rely upon the pseudoperoxidase activity of hemoglobin to produce a color change.

There are at least two problems with taking the positive result as strongly inculpatory evidence.  Problem one is that the ortho-tolidene test is subject to a number of false positives, and copper dust (common in the Chamberlain’s home of Mt. Isa) is one substance that produces false positive in catalytic tests such as this one.  Yet the head of the forensic laboratory downplayed the idea that the ortho-tolidene test gives false positives, claiming that interfering substances gave a different color than blood in this experiment.  When asked whether an experienced biologist could confuse a substance such as copper with blood in this test, he replied, “Not while I’m around.”  One wishes that someone had subjected Ms. Kuhl and her superior to a blind test of their abilities.

Problem two is related to the issue of substrate controls.  Substrate controls are used to check areas nearby a stain to see whether a positive test reaction is related to the stain or not.  Dr. T. Raymond wrote,  “The original trial scientist, Mrs Joy Kuhl obtained positive (ortho-tolidine) presumptive tests for blood in a number of areas in the Torana – including the carpet, bolt hole region, nearside seat hinge, console and items that were purported to have been in the car – a chamois, a towel and a pair of nail scissors. Some of these areas were incidentally inaccessible without the removal (using appropriate screwdriver) of surface panels.”  The inaccessible areas should have been considered substrate controls. The fact that they were negative positive should have given the forensic team pause but did not.  With respect to the luminol presumptive test for blood, Barni and coworkers wrote, “Regardless of how the stains are collected, the essential requirements to be met are the recovery of the available blood, the collection of a control sample in a tested area not exhibiting chemiluminescence and the complete drying of the support used for blood collection…”  Mrs. Kuhl also devised a test for fetal hemoglobin (Hb F).  The scientific questions surrounding the Hb F test are outside the scope of the present blog entry.

Gregory Taylor
 Mike Klinkosum wrote an article about the Gregory Taylor case in North Carolina, and how a number of cases of problematic blood forensics were uncovered in an audit. "What was not disclosed at Taylor’s trial was that NCSBI Agent Deaver had only provided the positive results from a presumptive test for blood known as a phenolphthalein [Kastle-Meyer] test. He had withheld from his official laboratory report the fact that he performed a confirmatory test (called a Takayama test) on the two stains that returned negative results for blood as to both substances...Agent Deaver had performed a third test on one of the suspected stains, a species origin determination test (referred to as the Ouchterlony test), designed to determine if the stain was human blood. He received a negative reaction on that test as well, but withheld the results of the Ouchterlony test from his official laboratory report."

Amy Driver reported on the aftermath of the Taylor exoneration. "Jill Spriggs, Chief of the Bureau of Forensic Sciences for the California Department of Justice and the President-Elect of ASCLD, responded [to a question at a hearing of the North Carolina state legislature] 'That is an accurate statement. A lot of times you got no results. It didn’t mean it wasn’t blood; it meant you didn’t have enough sample, or maybe the sample was old. …What else is red-brown that will give you a positive presumptive test for blood? There’s nothing that I know.'" This is a remarkable statement. Plant peroxidases are one source of false positive reactions, and rust is another.  With respect to most crimes, animal blood is also a false positive.

Mr. Klinkosum summarized four categories of wrongdoing with respect to how tests for the presence of blood were reported. He quoted the Swecker-Wolf report on the fourth category:  "The fourth and most serious category involves cases in which the reported actual results of the confirmatory tests were over-reported or not reflective of the results contained in the lab notes. There were five such cases in this category, all handled by [Special Agent Duane] Deaver.3 One of these cases involved a defendant who was executed. In two instances, the words 'revealed the presence of blood' were used when in fact the results of the confirmatory test were reflected in the notes as negative. This language was only used by Analysts when the presence of blood was confirmed by a positive confirmatory test. In three instances the report stated that further tests were 'inconclusive' or 'failed to give any result' when the lab notes reflect negative results. It should be noted that the Analyst, SA Deaver, advised reviewers that he was trained that confirmatory tests had only two possible results, negative or positive. SA Deaver’s lab files, however, revealed these two instances in which SA Deaver used the words ‘inconclusive’ in connection with Takayama test results despite his notes reflecting a negative result in one cases (sic) and three tests and three negative results in other cases."

The problems at North Carolina’s SBI laboratory go beyond one special agent, however. The Raleigh News and Observer reported, “Jed Taub, a 30-year veteran of the SBI, said, ’We didn't report the negative result of a confirmatory test because, really, it's misleading,’ said Taub, who now works as a forensic investigator for the Pitt County Sheriff's Office. ‘We couldn't be sure it wasn't blood, so those tests really didn't matter.’’ One might argue that Taylor's case is a different situation from the Knox/Sollecito case discussed more fully below. In the former case, a negative confirmatory result was withheld. In the latter case, the TMB results were withheld until late in the first trial. However, the more general principle in question is whether or not forensic scientists should disclose all test results and let the chips fall where they may.

In one of an award-winning series of articles at the Raleigh News and Observer, Joseph Neff and Mandy Locke wrote, “The phrasing of State Bureau of Investigation lab reports from the 1980s to 2003 caused a lot of ruckus in 2010, raising the question of whether North Carolina's practices that helped prosecutions were common to crime labs across the country.”  The executive summary of the 2009 National Academy of Science report “Strengthening Forensic Science in the United States: A Path Forward” noted on p. S-15, “The terminology used in reporting and testifying about the results of forensic science investigations must be standardized.” It is unfortunate that the NC SBI laboratory did not adhere to this principle in the Taylor case.  "Regardless of how everyone else did it 20 years ago, the current and best practice is to report the results of all tests, just as the [North Carolina] SBI lab does now," [SBI Director Greg] McLeod said.  Director McLeod’s comments are suggestive of an improved culture at the SBI lab.

Laurence Lovejoy
The Laurence Lovejoy case from Illinois involves the lack of disclosure of a presumptive test. The Illinois State Supreme Court Granted Mr. Lovejoy a new trial. "Despite the positive LCV [leucocrystal violet] test, Camp tested the swab with TMB, a more sensitive and more reliable presumptive test for the presence of blood, as her protocol dictates. The TMB test was negative, indicating that the substance swabbed was not blood." The opinion goes on to state, "The parties do not dispute the facts giving rise to the alleged discovery violation. Both sides agree that Camp did not include her finding that the TMB test produced a false negative, and did not state her reason for the negative TMB test in her report."

The opinion also stated, "Further, if defendant were made aware of Camp’s conclusion that the TMB test produced a false negative because the LCV used all the hemoglobin in the blood, he could have called an expert to refute this contention during his case in chief, or could have chosen to pursue a different line of defense altogether. Defendant was prejudiced when Camp’s conclusions were revealed for the first time at trial." Earlier in the opinion it was made clear that such an expert indeed existed: "In that motion, defendant stated that Dr. Karl Reich, who holds a degree in molecular biology from the University of California, Los Angeles and Harvard University, was prepared to testify that the TMB test used by Camp is the most sensitive presumptive blood test available; that a negative TMB test strongly suggests that there is no blood in the area tested; and that Camp’s testimony that the LCV consumed the reactive blood components, thus confounding the TMB test, was incorrect."  In other words, not knowing that there was a negative TMB test nor knowing what rationale would be offered for its being negative hampered the ability to present a robust defense.

Amanda Knox and Raffaele Sollecito
The luminol-positive areas in the Knox/Sollecito case include two blobs in Filomena’s room, three bare feet in Amanda’s room, three bare feet in the corridor, and one shoe print in the corridor.  The two amorphous shapes in Filomena’s room and the shoe print have Meredith and Amanda’s DNA profile, the three prints in Amanda’s room have her profile, and the three bare prints in the corridor have no DNA profile.

A number of forensic references recommend the use of a colorimetric reagent such as TMB on areas that are luminol- or fluorescein-positive. Lisa A. Gefrides and Katherine E. Welch wrote,  “Either luminol or fluorescein can be sprayed onto large surfaces such as walls or floors and the positive areas areas marked for further testing. Both tests are very sensitive and will indicate bloodstains that may not be visible…One disadvantage to these tests is that both can have false positive reactions. Luminol and fluorescein will react with the same false positives as PH [phenolphthalein] and also with bleach and other cleaning fluids, which may interfere with blood detection on surfaces that have been cleaned. For this reason fluorescein or luminol positive areas should be retested with one of the color change presumptive tests.”  (“Serology and DNA” in “The Forensic Laboratory Handbook Procedures and Practice,” Humana Press, 2006) Stuart H. James and William G. Eckert wrote, “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.”  (p. 162, “Interpretation of Bloodstain Evidence at Crime Scenes,” 2nd ed., CRC Press, 1998)

A technical bulletin on Lightning Luminol said, “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…A second presumptive field test should be used on the areas that were detected positive with Luminol. Results should not be reported as a positive presence for blood, only positive indications of blood. It is recommended that laboratory testing, if possible, be followed on staining areas detected at scenes.”  In “The forensic luminol test for blood: unwanted interference and the effect on subsequent analysis,” Anders Nilsson of the Swedish National Laboratory of Forensic Science wrote, “Due to the lower selectivity of the luminol test positive reactions should be verified by other presumptive blood tests like the phenolphthalein test [James, S.H., Kish, P.E. and Sutton, T.P., Principle of bloodstain pattern analysis: theory and practice, 2005 CRC press Taylor & Francis Group, ISBN 978-1-8493-2014-9].”  Although one group of workers (“Accuracy, Reliability, and Safety of Luminol in Bloodstain Investigation,” Canadian Society of Forensic Science Journal 35 (3), September 2002, 113-121) has suggested putting an end to the practice of following luminol with a colorimetric test, it would seem that many laboratories do use the TMB or the Kastle-Meyer test on luminol-positive areas. 

The fact that some or all of the luminol-positive areas had been tested using TMB with negative results did not emerge until Sarah Gino’s testimony in September of 2009, near the end of the first trial. The Massei report stated, “With respect to the Luminol-positive traces found in Romanelli's room, in Knox's room and in the corridor, she stated that by analysing the SAL cards ‘we learn, in contradiction to what was presented in the technical report deposited by the Scientific Police, and also to what was said in Court, that not only was the Luminol test performed on these traces, but also the generic diagnosis for the presence of blood, using tetramethylbenzidine...and this test...gave a negative result on all the items of evidence from which it was possible to obtain a genetic profile…’” (pp. 256-257, English translation)

There has been much discussion about whether or not the luminol-positive evidence items in the Knox/Sollecito case are blood.  I will outline six reasons to be skeptical of the notion that the greater sensitivity of luminol is the explanation for why the TMB test was negative for luminol-positive areas in this case:

There are substances in some of the photographs of the application of luminol that give off a blue glow, as posted in these threads. There were blue flecks on a ruler, on a pair of boots worn by someone within the forensic police, and on the grouting between tiles.  The technical bulletin for Luminol Lightning noted that, “Luminol can give a low grade reaction with some carpet materials.” False positive reactions are seen with many substances besides bleach.

The technical notes for Luminol Lightning caution, “Also, tracking through an area that has been sprayed with Luminol will produce brighter shoe tracks. These tracks can be transferred to other parts of a crime scene. Care should be taken not misinterpret these reactions.”  This might explain the luminol-positive areas in Filomena’s room; however, whether the hallway or Filomena’s room were sprayed first is unknown.  Alternatively, one can speculate that the luminol-positive areas in Filomena’s room were the result of foot traffic carrying an unknown substance between 2 November and 18 December, when the luminol was applied.

Ranges of values for the sensitivities for both the luminol test and the TMB test have been reported in the forensic literature. Some values suggest that luminol test slightly more sensitive, but I have seen claims that both tests can detect blood that is diluted up to 1 part in 1,000,000. However, let us assume for the purposes of illustration that TMB detects blood to 1 part in 10,000 and that luminol detects blood to one part in 100,000, a tenfold difference in sensitivity. However, there is a 10,000 fold range (from undiluted blood to blood that has been diluted ten thousand fold) in which both will be positive. In other words each and every dilution would have to be between 10,000- and 100,000-fold to be TMB-negative and luminol-positive in this hypothetical situation.

Some of the luminol-positive areas were also negative for DNA.  Luminol has a slight negative effect on how much DNA can be culled from a sample, and this depends on the formulation. However, it is certainly not the case that the use of luminol precludes testing for DNA. Moreover some luminol-positive areas did have Amanda’s DNA, so we know that luminol does not always interfere with DNA profiling. So if the luminol reaction were responding to Meredith’s blood, why is there none of Meredith’s DNA in some items of evidence and no DNA at all in others?

If the only reason why TMB would be negative when luminol was positive is the lesser sensitivity of TMB relative to luminol, then there would be no reason whatsoever to follow a luminol test with a TMB. A negative TMB would not be meaningful, and a positive TMB is still not a true confirmatory test. According to the Massei report (p. 258, English translation), Sarah Gino testified about the use of TMB: “She added that, in her own experience, analyses performed with TMB on traces revealed by Luminol give about even results: 50% negative, 50% positive…” This observation suggests that the purpose of the TMB test in the field is to reduce the number of false positives, as also seems to be true based on the citations above. This interpretation is also consistent with Stefanoni’s testimony about the nature of the TMB test, as discussed in the Hellmann report: “Professor Tagliabracci, specified, without being refuted (hearing of July 18 2009, p. 174), that the tetramethylbenzedine (TMB) test is very sensitive, so much as to give a positive result even with only five red blood cells present. Dr. Stefanoni herself, moreover, clarified (preliminary hearing of October 4 2008) that, while a positive test result could be deceptive due to reactivity of the chemical [evidenziatore] with other substances, a negative result gives certainty that no blood is present.”

As blood is made more dilute, the luminol reaction becomes fainter. There is no evidence to suggest that these luminol reactions were especially faint.

A theme in at least three of these cases is that the forensic personnel have an implicit or explicit belief that a positive result in a presumptive blood test means that blood is really present and a corollary belief that any negative results in subsequent presumptive or confirmatory testing is the result of a failure in the follow-up test.  Putting it another way, the Knox/Sollecito case is not unique with respect to its misinterpreted presumptive tests for blood.  It is an open question whether or not the false beliefs would change if forensic laboratories were made more autonomous from prosecutor’s offices or law enforcement.  This is recommendation 4 in the 2009 National Academy of Science report “Strengthening Forensic Science in the United States: A Path Forward.”  Both the full report and an executive summary are available.

In the Chamberlain case, personnel from the forensic laboratory made statements about presumptive test that were false.  They destroyed plates that involved the HbF testing before the defense had a chance to examine them.  In both the Taylor and Lovejoy cases, the state's failure to disclose the test results was simply wrong.  Likewise in the Knox/Sollecito case it was wrong for Ms. Stefanoni to fail to disclose her negative TMB results prior to the trial.  Even when these results become available to the defense during the trial, this may be too late for the defense to adapt its strategy to the new information.


RoseMontague said...

Wow, just wow.

You put it all together Chris. Great job!

Dougm said...

Great job, Chris! Really well done.

Sarah said...

Great article Chris, very interesting.

Anonymous said...

Thanks Chris!

Paul Smyth said...

Very well done. I hope that if any of the pro-guilt faction from the Knox-Sollecito case show up they will be so good as to give us their real name and professional qualifications--as you have done Chris.

Anonymous said...

The point is that even if it was DNA and not blood from Amanda Knox so what.With DNA combined with MK's blood to form the 'blobs' how did it come to mix together? Did not the trial judges think that shed DNA from the suspect could have mixed during hand washing to remove blood? If it were tracked on a boot cover as you suggest there would have been a fading trail.A flick off a washed hand is more believable. Paul common sense is a great credential.

Rick Bonin said...

Anonymous - Amanda's DNA will be all over the apartment, as she lived there. Common sense, remember?

Chris Halkides said...

Anon. at 10:40, Amanda's DNA in her own bathroom is one of the most unsurprising of all results in this case, as I pointed out in my entry on mixed DNA last year. Meredith's blood may have landed on Amanda's DNA that was already present. Another possibility is that Meredith's DNA and Amanda's DNA were is close proximity (for example in the bidet), and the act of sampling over such a wide swath mixed them. A third possibility is that the failure to change gloves after every sample resulted in mixing of DNA. Mixed DNA is a common phenomenon. Massei made a conjecture about DNA deposition during the cleanup, but it was just speculation. What is your explanation for the blobs in Filomena's room?

Anonymous said...

I think that the blobs came from someone involved in the murder. It had to be someone who went into Filomena's room after the fact. If they were tracked on a shoe cover( pure speculation ) there would have been a fading trail. I think the blob could have come off of a hand that was freshly washed. The blobs in Filomen's room cannot be explained away by cohabitation.It could have just been a drip from an opening hand. The murder had happened and the suspect had gone back into the room.Guede had no reason to return there.I thought I read somewhere that Filomena was only missing makeup? You would think that Guede's DNA would have been shed in the bathroom or on the blob if he were the one who had scrubbed up so vigorously. Who ever tossed the phones and locked Meredith's door had cleaned off all traces of blood on their hands.

Paul Smyth said...

This is really getting silly anonymous. You seem to be forgetting that the burden is on the prosecutor to demonstrate that a finding has some relevance to the murder, something the clowns Mignini and Comodi were spectacularly unable to do. The mixed DNA in itself proves nothing since there is absolutely no evidence that it was the result of anything other than being roommates.

Again, Chris is a Ph.D. biochemist whose name is known to us al. And just who are you and what are your credentials?

Anonymous said...

They did find something, DNA mixed with blood in a few random blobs. Those blobs are huge as far as evidence goes. They were in Filomena's room, and were deposited after the murder and cannot be explained by cohabitation. I have all the credentials you need to find that hard to explain Paul.

Chris Halkides said...

The luminol-positive blobs in Filomena's room were tested by TMB, which was negative. Apparently, they were never tested with a confirmatory test; therefore, they cannot be concluded to be blood. One can make up stories to the effect that someone put his or her hand down, but that kind of story is just conjecture. If luminol did not give false positives, there would be no reason to use TMB and a confirmatory test.

The luminol positive areas should have been subjected to substrate controls. That might have strengthed the case that they were probative.

The luminol story comes apart once one considers Rep. 180, a luminol-positive footprint in Amanda's room. The second toe is much shorter than Amanda's. This print must be from a barefoot woman, yet there is no woman besides Amanda in the prosecution's scenario. Therefore, the print is unrelated to the crime. There is no reason to assume that this print must be blood (in fact once one realizes that the print could have been made at any time, the chances that it is blood drop even further). If this print were not made in blood, then there is even less reason to assume that the hallway footprints were made in blood. Moreover, the existence of Rep. 180 forces one to ask whether this unknown female also made a print in the hallway.

Chris Halkides said...

Anon. at 9:50, You wrote, "You would think that Guede's DNA would have been shed in the bathroom or on the blob if he were the one who had scrubbed up so vigorously." With the tap running, there may have been little or no DNA deposition. There is no way to date DNA or luminol; therefore, any positive reaction could be the result of things that happened either before or after the crime.

Anonymous said...

Chris you are talking about blood and putting one's hand down, nothing I mentioned. I think that the isolated blobs of blood and DNA were deposited by someone after the murder and show no evidence of being tracked in by foot.Are you are saying or believe that the blobs contain no evidence of AK? Simple explanation please as my credentials are limited to common sense.

RoseMontague said...

Chris, great point about the print in Amanda's room. The two blobs (still no pictures) in Filomena's room are also a mystery. It is unknown what made them, when they were made, or if they are even related to the murder.

The two shoe-prints (revealed by finger print techniques) on the envelope and postcard present on the rug near Filomena's bed however are subject to common sense reasoning. Both of these reveal a completely different pattern than that of the shoes of either Rudy, Amanda, or Raffaele. One is a small zig-zag and the other is of small circles, not the large rings found in Rudy's or Raffaele's shoes. It looks like they were either knocked of the bed or the nightstand at the time of the break-in and someone stepped on them at a later time, leaving those prints. I doubt someone stepped on them as they lay on the bed or nightstand.

My guess is that they were made by Filomena and possibly other people that came into that crime scene without protective footwear who stepped on them as they were walking about destroying the nature of the evidence in Filomena's room.
There is no telling what else was dragged in there that could have been deposited on a spot already there (or not). Like the glass on top of, in the middle of, and under clothes, the evidence in Filomena's room was compromised.

Chris Halkides said...


You raise a a point that does not get made frequently enough: By 18 December the crime scene had been horribly compromised by all kinds of traffic and moving of objects.

I corrected a typographical error just now. The incorrect word is now in strikethrough.

Kaosium said...

Chris, I would never have thought I'd find this subject so fascinating, thanks (mainly) to you I did! :)

Anonymous, I suspect the problem here is you're still referring to the luminol hits as 'blood' despite the negative TMB tests, that's quite unlikely actually, it's a natural chemical reaction that occurs, if it doesn't occur then the substance *isn't* blood, outside some unlikely probabilities. That they advanced such unlikely probabilities only when they were caught not disclosing the negative TMBs does not make them inherently valid--it makes them suspicious.

There's no corroborating evidence it occurred that way, it's just a 'hypothesis' they came with. When considering the likelihood that somehow Amanda managed to get into Meredith's blood as a result of being involved in the murder yet managed to leave such perfect uniformly diluted prints (without design!) is perhaps the most implausible thing about the case outside that knife ever leaving Raffaele's drawer that night.

I mean, how did this occur, did she stir this mixture of highly diluted blood in the water like a drink? Remember the prints (that they disclosed pictures of) all look pretty much bright and bubbly throughout both the print itself and from each to the other, how did that occur with such severely diluted mixtures of blood and (presumably) water that despite several occurrences in the cottage in (sometimes) wildly different locations without a path they all fell between the threshold between TMB and luminol yet managed to shine so bright for the cameras?

Let's pretend for a minute there's a rational possibility it all happened that way, I can't think of how (and I have a vivid imagination!) but I will simply postulate that--now what was the source of this blood? How did Amanda manage to get it on her without having left any trace in the murder room and where did it become so diluted in a way that it most probably implicated Amanda in the crime and didn't just 'prove' that Amanda was walking around the cottage hours before the discovery of the murder, something already known long before the discovery of the prints six weeks later?

In a sense that's the most damning implication of the luminol hits and the integrity of Stefanoni, even if those prints were highly diluted blood, there's an obvious innocent explanation for their existence: Amanda stepped in a film of water where a minute amount of Meredith's blood (either because of the murder or previously during the course of her living there and bleeding on a monthly basis) and tracked that back to her room when she was done with her shower at any time they were living there!

Kaosium said...

That's the thing: there's nothing to connect that 'diluted blood' to the murder room anyway, nor any reason to think a 'mixture' of blood that diluted had anything to do with the murder regardless. Even if Amanda had gotten some minute amount of blood on her, she was padding around a crime scene where Meredith's blood was present (even reported it to the police!) and a tiny amount like they 'hypothesized' doesn't 'prove' anything outside maybe she got some on her while walking around a crime scene--where she lived.

You know there were bloody tissues found outside the cottage, right? Were they necessarily involved in the murder? None of the DNA matched anyone they knew, is it 'proof' that perhaps Rudy was telling the truth to Alessi or about the 'left handed Italian' when first asked about it? Or is it quite possible those tissues were from another incident entirely unrelated to the murder and more so to the denizens of the drug culture as represented by the likes of Curatolo?

The only thing the luminol hits 'prove' is how fundamentally dishonest and desperate the prosecution was: even if that was Meredith's blood in such a diluted amount there's no reason to think that was the result of anyone being involved in the murder anymore than there is to think there were two or so other males outside Raffaele involved because of the DNA 'found' on the bra clasp, or those tissues were 'evidence' Rudy must be telling the truth.

However, if Stefanoni wasn't being dishonest, explain to me in small words I can understand just why she didn't do a confirmatory test on them? These presumptive tests just suggest they might be blood, the proof is in the confirmatory test, why on earth would she fail to prove that was blood in Amanda's footprints? Why wouldn't she perform the test that would actually prove her 'hypothesis' was true? She employed the 'species specific' (confirmatory) tests on other items, why would she not perform this test on what would appear (on the surface at least) to the average person to be such damning 'evidence?'

Because it wasn't blood, just like the TMB tests said, all the rest is just Stefanoni's imagination. You do realize that, don't you? There's no evidence the blood was diluted outside that it failed the TMB test. Well, except perhaps by not performing (or admitting to it if she did do them and didn't like the results) the confirmatory tests she pretty much revealed that she had no interest in doing anything but deceiving the court.

Think about that for a moment. She had the opportunity to prove it was blood but chose not to perform the routine test that she did for other samples (some positive, some negative) but claimed she never did one for these 'bloody footprints' which on the surface would seem by far and away to be the best evidence Amanda was involved in the murder? Uh, why would that be?

Because they weren't blood, and even if they were diluted to that extent they certainly weren't evidence of much of anything outside that Amanda was walking around a crime scene before the murder was discovered and didn't know it because the traces were invisible.

It doesn't actually matter much either way, but the TMB tests are the best evidence of what they were: something other than blood, quite probably a cleaning product of some sort considering their origin--which was not the murder room, now was it? They go from the bathroom to Amanda's room, whatever made you think they were involved in the murder anyway, outside the prosecution saying so?


Anonymous said...

All of these tests are overrated. For example, it has been shown by Morgan Cheyne that luminol destroys DNA. How many of the investigators know this? How many of investigators have been provided adequate training? How many of these investigators even know how these tests work? How many of these investigators even have degree in biology or chemistry? We need more training and hiring credentials. It is organization's fault to not provide enough training. It is not a stretch to say that eveidence based on such tools will be often discredited by defense.

Chris Halkides said...

Anon. at 10:19,
Do you have a citation? I have read a little bit about extracting DNA from luminol samples, and my recollection is that the amount of DNA goes down slightly, but one can still obtain a profile. I have also read that it depends on the particular formulation of luminol. The review article by Barni and coworkers has some citations on this subject. I don't have a copy of this reference yet, but the abstract is a good beginning. A study by Gross and coworkers does not support a strong adverse effect of luminol on DNA analysis. I don't know which formulation of luminol was used in this case.

Chris Halkides said...

Anon. at 10:19, I completely support reforms which would see that forensic workers are better trained, not just in their disciplines, but also in the scientific method more generally. Either their training is not always what it should be or the culture of the organization does not encourage free enquiry, as both this entry and my previous entry on presumptive testing documents. With respect to this case, the quality of the forensics is the main point of contention, because nothing else supports a guilty verdict.

Anonymous said...


Anonymous said...

1) So you contend that there is no reason to think that the Luminol positive result was showing a positive for blood. Could you remind me what it could be showing a positive for? After all, if the list is small we might be able to consider some of them to be unlikely. I believe fruit juice is one of them. Would a fruit juice spill explain the footprints? The distribution and location of the footprints might not make very much sense assuming another substance. Also would cleaning with bleach explain the difficulty in obtaining a positive test for the presence of blood?

2) Wasnt there copious luminol traces of blood in the bathroom? I get the impression that you are not disputing luminol traces in the bathroom. If I have that right, that would mean that someone cleaned the bathroom, and made a bloody print on the bathmat but the footprints revealed by luminol are not related to the blood and it is just a coincidence.

I find this implausible. Your theory that the footprints were not caused by blood appears to be possible but not plausible.

If you have the time, I would appreciate your take on this. It is quite possible I am totally mistaken.


Chris Halkides said...

Anon. at 6:08, There are three left footprints in the hall. If they were made in blood, where are the right feet? Bleach, draino, and some other cleaning products give false positives, as well as certain fruit pulps, rust, and certain transition metal ions. Also, animal blood would give a positive result, and I don't intend this list be taken as an exhaustive one. The most central problem I have with your first comment is that one must obtain a positive confirmatory test to conclude that it is blood. Your comment suggests that it is up to the defense to show what the luminol-positive substance was, and that is contrary to general forensic practice.
2) There were no luminol-positive spots in the bathroom. There are no streak marks in any photos I have seen of the luminol traces, which disfavors the notion that there was a clean-up.
3) How do you explain Rep. 180, the luminol-positive footprint in Amanda's room?

Anonymous said...

The luminol testing should NOT be accepted in court for many many reasons. It is flawed forensics. Many of the technicians doing luminol analysis do not even have high school diploma or understand the science behind it. Every prosecutor and defense attorney should question the results from it and DNA results from them are so variable that they are totally unreliable. I have heard the argument that swabs are already taken for DNA before application as there is enough literature out there that luminol destroys DNA depending upon formula used, how much is sprayed. How can you take samples of blood when you do not know where it is?


“A scientific article about luminol available here notes that it can react with components found in "soils, detergents, bleaches, carpet, metal objects, tools, plastic panels, wood, and vegetable compounds." 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 article linked above notes that, because luminol reacts with many non-blood substances that may be present at a crime scene, "the luminol test must not be considered sufficiently specific to permit an unequivocal identification of blood."

Anonymous said...

How does one contact prosecutors in Amanda Knox case? is there an email?

Chris Halkides said...

I don't know how to contact them.

Anonymous said...

First of all, very kind of you to answer. Very much appreciated.

"Your comment suggests that it is up to the defense to show what the luminol-positive substance was, and that is contrary to general forensic practice."

I did not mean to imply that at all. Instead I am trying to evaluate the likely scenarios which might have led us to see the evidence we see.

To summarise, there are luminol footprints in the house. They do not test positive for blood but apparently if they were cleaned then its possible to get a positive test result for luminol and a negative result for blood, even if it was caused by blood.

These prints are in discrete locations. They could have been caused by exposure to bleach, human blood, animal blood or fruit juice. Or other items. But we know that they resemble footprints so we know that they must have trod in the material and made a few steps in it cos we have distinct footprint in the luminol. However we are missing the half the foot prints and we are also missing the connecting foot prints.

So we could have had one of the young ladies step in animal blood and leave those prints a few weeks earlier. Or fruit juice. Personally I dont see how they could have trod in an area of rust and left only a few imprints but I guess its possible. Or the print might just be human blood. After all, it is a murder scene. And there is quite a lot of blood around. And there is a bloody footprint on a rug in the bathroom.

So for me, the odds imply that it is probably blood. But its not impossible that it isnt.

I am not suggesting that the defense has to prove it isnt blood. Im suggesting that we should only consider reasonable scenarios in looking for reasonable doubt. The combination of events the defense asks us to believe is problematic.

To name just three,

1) That the traces of DNA on the knife blade was the result of lab contamination.

2) The DNA on the bra catch was collection contamination.

3) The luminol footprints are fruit juice.

Any one of these 3 is possible. All three together are problematic. Unless one assumes a deliberate effort to incriminate on the part of the forensic lab.

Also, I got the impression that there was a photo taken which was suggestive of a substantial clean up in the bathroom particularly of the sink. I shall have to look that up cos I cant find it in the judges report.

Thanks again for your reply.

Chris Halkides said...

Metal ions might be found in soil, and a number of cleaning agents produce a false positive (not just bleach). Both the negative TMB and the lack of DNA are suggestive that the luminol prints are not blood. In addition, there is no confirmatory blood test, and the luminol footprints don't show any streaking. With respect to the bathroom, you may be thinking of the pink bathroom photo, which I discussed in a previous entry. My interpretation of that photo is that it is a failed Kastle-Meyer test; however, this photo was never entered into evidence, so one cannot say with certainty.

One problem with the luminol footprints is how to account for only left feet (and one seems to point toward Meredith's room, not away from it, IIRC). However, another problem for the prosecution is the presence of a luminol footprint in Amanda's room which is clearly not Amanda's footprint, on the basis of the second toe. A woman made it, yet the prosecution's case does not involve any other woman. The print was likely made before the murder; therefore, there is probably a luminol-positive substance that is not blood that was responsible.

Chris Halkides said...

Since the last time I wrote about the knife, I have read more about bleach and DNA, and that reading makes me even more inclined toward laboratory contamination, not Meredith's blood, being the source of the DNA. The knife peaks, although varying in size, show no sign of degradation.

Anonymous said...

It would be wrong to say that I have not come to a provisional conclusion. However I very much hope I remain open minded enough to listen to other people's reasoned argument. So I will look out for your piece on why you think the lab results are very likely contaminated. But it seems to me that in asserting contamination, the burden of proof is on the defense and not the prosecution. After all, they had the opportunity to attend all the tests and declined. If they had attended they would be better placed to suggest problems with the nature of the test.

Still there is one piece of evidence that seems inconsistent with your argument. We have one bloody footprint. To my knowledge no one has disputed that the footprint is related to the murder but perhaps you do. Assuming you dont, you will agree that someone must have trod in blood in bare feet at some point. And then stood on the mat. The pool of blood would be in Meredith's room.

So why are there no other bloody foot prints? How did that one footprint get there? Are you suggesting that Guede took his shoes off - got blood on his foot - cleaned them off in the bathroom -and cleaned up any other bloody footprints - and then put them back on to escape the scene of the crime?

It just doesnt make sense to me but I may well be missing something obvious. If I am right, this is strong presumptive evidence that someone else was present. Someone who got blood on their bare feet. Who, is open to debate.

And once again, thank you for engaging with me. I am very grateful for the opportunity to test my thinking with someone who has looked at this question in some depth.

Chris Halkides said...

I don't have time to address all of your comments at the moment. Briefly, I have heard two reasonable theories about the bathmat footprint. One is that Guede made it after cleaning his trouser leg in the bidet, and the other is that he made it after doing so in the shower. He may have stepped onto a towel over the mat not realizing that some bloody water seeped through.

With respect to the DNA testing, my entries on the electronic data files make clear that observing the testing is nice, but it is not nearly as important as having access to the files. Thus the principle of discovery was violated. Even Conti and Vecchiotti complained about this.

I have twice referred to Rep. 180, the luminol print in Amanda's room, in these comments. What is your theory of how and when it was made?

Anonymous said...

I have split my reply in two.

So first of all I am grateful for any time you choose to spend
debating this with me. There is no need for you to address any of my
points or to use time you don’t have. I am still grateful you debate these questions at all.

Secondly, let me address Rep.180. I havn’t read much about this piece
of evidence so I shouldn’t comment. However I don’t understand why you are quite so confident that the footprint couldn’t have been from AK. It occurs to me that perhaps the surface area of the foot was not uniformly covered in fruit juice or whatever other substance. I have read your piece above, but I didn’t fully understand why this print could not possibly be Amanda's. I really need to go back through your prior posts.

It was reported at the trial that Amanda Knox’s lawyer, Luciano Ghirga, asked Dr. Stefanoni to confirm that other substances like bleach or fruit juice can also react to luminol. Dr. Stefanoni acknowledged that they do, but pointed out that biologists who work regularly on crime scenes distinguish easily between the bright blue glow of a blood trace and the much fainter glow from other reactive substances. This doesn’t directly address your point, but it does suggest that the Italian forensics are aware of the potential for confusion.

However your comment reminds me about another piece of evidence. A bloody shoe print found on the pillow below Kercher's body.

“When the judge asked Rinaldi the size of an unidentified bloody shoeprint found on the pillow below Kercher’s body, he responded, “Between 36 and 38.” The judge then asked Rinaldi what size shoe Knox wears. “The Skecher shoe we sequestered belonging to Amanda Knox corresponds with size 37.” Lots of ladies must have size 37 feet. But we can say that there probably was a female in the apartment during the murder. And she left a shoe print. The same woman might have left the Rep 180 evidence too. But clearly I am speculating.

I think you would be on stronger ground with that conclusion if the
print was too big rather than too small. Too small or missing some
element does not rule out the possibility that the print belonged to AK. Too big would. Like the argument presented in the court that Guede’s foot was too narrow to be a match for the print on the rug and that RS’s was a better fit.

Anonymous said...


Two footprints were attributed by the prosecution to Raffaele Sollecito. One of them was revealed by luminol in the hallway, and the other one was easily visible to the naked eye on the blue bathmat in Meredith’s and Knox’s shared bathroom.

Lorenzo Rinaldi excluded the possibility that the bloody footprint on the blue bathmat was the right size or shape to belong to Knox or Guede instead of Sollecito: “You can see clearly that this bloody footprint on the rug does not belong to Mr. Guede, but you can see that it is compatible with Sollecito.”

I have read that Rudy Guede’s visible bloody footprints lead straight out of Meredith’s room and out of the house. Do you know whether this is true? I ask because it was not from a primary source and I have learned to be suspicious of anything which I did not read in the Judges' reports. If this is incorrect I apologise and I am grateful to be corrected. If this is correct then it would make it very difficult to assert that Rudi left the bathmat print.

I am not a forensic scientist so I have no useful comment on Conti and Vecchiotti. I leave that to the qualified.

Now as regards the bathmat footprint theories you suggest (which are the most interesting bits for me). So the scenario is that he had blood on his
clothes, which he was washing out. This was very brave of him
considering that he had no idea when Meredith’s room mate might get
back. He took his shoes off to clean his trousers? That’s really
nerves of steel in the circumstances. And if he was so worried about leaving evidence, perhaps he would have been well advised to flush the toilet in the other bathroom? Why switch bathrooms to use the little one when you have an awkward job to do. Why not use the first bathroom that you went into? Its not like he was familiar with the flat.

Was there a bloody towel at the crime scene? If not, where did it go?

What did he clean his clothes with? Did he leave DNA behind in that bathroom? I havnt read that he did. Was there any shoe print in the bathroom? Not to my knowledge. Unless he cleaned up after himself then it doesn’t seem sensible to suggest that RG left the print on the bathmat.

The theories you suggest for the bloody bathmat make much more sense
for someone other than Rudy. I would suggest that whoever left them felt comfortable cleaning up at a relatively leisurely pace. Probably cos they knew no one was coming back to the flat soon.

But you are right. The existence of the bloody footprint on the mat suggests that someone did some cleaning in the bathroom. Odd that they found no DNA in the bathroom other than that of the flatmates.

Anyway, I have my doubts and I have explained some of the reasons why. I am confident that there was more than one killer. I am confident that someone cleaned up at the scene and that someone moved the body from the its initial position. I strongly suspect the break in was staged, and I am concerned about inconsistent statements and the lamp found under Meredith's bed. But I am just one person and I can be wrong. Thank you for your efforts in seeking the truth.

Chris Halkides said...

Anonymous, Amanda's second toe is longer than her big toe, but Rep. 180 shows a foot for which the opposite is the case. I have not addressed this item in my posts yet, but it has been commented upon at Websleuths and JREFF. The article by Barni and coworkers on luminol note that some forensic workers believe that they can tell luminol from other substances (not just bleach or fruit juices, BTW); however, this article suggests follow up testing to minimize subjectivity.

With respect to the print on the pillow, Professor Vinci's presentation (which can be found at Injustice Anywhere) discusses this (in Italian). Although I did not have a human translation, I looked at the photos and used a machine translation. My understanding is that there were two parallel lines that Rinaldi chose to interpret as the outer edges of shoe print, in other words, a partial print. However, there are no parallel edges on any shoes that Vinci showed, and I have looked at some pairs myself. Vinci believes that the two parallel lines are actual one edge that transferred some blood to another part of the cloth by folding and blotting. Please let me know if this is not clear.

Rose said...

Massei chose not to side with Vinci or Rinaldi on this disputed shoeprint on the pillow, citing his belief that Amanda was barefoot.

Anonymous said...

Thanks for your answers (and Rose). Much appreciated. I shall dismiss the shoe print from my mind. Could you also help with whether there really were visible bloody footprints assumed to be Guede leading out of the house. I have read that, but I dont remember seeing it in the Massei report.

Clearly if its true it would be important as it would suggest Guede didnt leave the bathmat print.

Is there anything else I wrote that you think is wrong, or wrong headed? Im not sensitive. I just want to clear up my thinking on this question.

The DNA in the bathroom? Was anyone elses DNA discovered there?

Chris Halkides said...

Rinaldi's work deserves to be judged in its entirety. Not only did he get the print on the pillowcase wrong, but he also worked with reference prints only from Raffaele, Rudi, and Amanda, not the other flatmates or anyone else. This is a good example of tunnel vision. The experts who worked on the defense obviously disagreed with Rinaldi. I have looked at Raffaele's reference print, Rudi's print, and the print on the bathmat. I don't think that it should be attributed to any individual. However, to my nonexpert eyes, it looks more like Rudi's than Raffaele's, particularly in the big toe.

Guede's shoe prints get fainter toward the door. IMO he stepped in blood when he returned to Meredith's room to take her cell phones and other items. In other words, I think he made them after making the bathroom print. The prosecution's first shoe investigator attributed those prints to Raffaele, which was a foolish mistake.

Chris Halkides said...

The bathroom was only shared by Amanda and Meredith; Laura and Filomena used the other bathroom. I think that this, along with poor sampling technique, explains the mixed DNA. However, the question always arises why is Rudi's DNA not there. If he simply washed his hands, that might not leave much DNA. I am not sure whether or not he was bleeding that night. However, if he were, the blood was probably on the towels that he took. Unfortunately, the towels were not stored properly. I seem to recall that they did not yield any DNA.

Chris Halkides said...

I think Rudy may have forgotten about the feces in the other bathroom. I think Rudy was balancing the need to remove enough blood from his clothing so that he would not arouse suspicion against a concern about how long he was taking. That may be why he did not attempt a more thorough clean up.

Anonymous said...

Thanks you so much for your candid replies. I feel I have a much better understanding of both sides of this debate. It is very good of you to give up your time for this.

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Anonymous said...

Thank you for this extensive explanation of this area of forensics. I had no knowledge of this at all, I think I am like many people, simplistically assuming these tests give simple yes or no answers and the lab people are doing honest objective work.
It's funny that I assumed that, because I don't think other technical questions are typically yes or no. But the media encourage shallow thinking, and it's easy to fall into it.

Your blog is the best thing I've every read to explain this, and particularly pointing out the test labs under prosecution control assume some test results are good and disregard those that work against guilt.

Chris Halkides said...

There was a report by the National Research Council called, "Strengthening Forensic Science in the United States: A Path Forward." IIRC they suggested splitting forensics labs away from law enforcement. I don't think that this would be a panacea, but I think it would help.

Geysi Castillo said...

Wow, it is amazing how some professionals don´t do their job in the correct way. I´m studying criminal investigation and other thing I consider is very important, is that they stablish that footprints were made with blood, but if they were really blood, the luminol wouldnt give a footprint, it would be a footprint that wanted to be cleaned