Thursday, September 19, 2013

The Italian supreme court’s dangerously erroneous views on forensic DNA contamination

Part 37 in the Knox/Sollecito case

Part A:  Errors in the principles of DNA forensics and discovery
The Italian Court of Cassation, the supreme court of Italy, released a report giving its reasons for overturning Amanda Knox and Raffaele Sollecito's successful 2011 appeal and sending the case for another trial in Florence beginning at the end of September.  The report by the Corte Suprema di Cassazione (CSC) has been translated into English twice, and a comprehensive critique has appeared.  Page numbers below refer to the first translation.  The CSC’s report made a number of highly questionable assertions or omissions with respect to DNA contamination in general and with respect to the evidence in this case:

“It was also ruled out by the same experts that contamination occurred in the laboratory. Professor Novelli said that the origin, the vehicle of contamination must be demonstrated: he specified to have inspected 255 forensic sample extracts at the Polizia scientifica, had analyzed all profiles and did not see any evidence of one single contamination; he excluded absolutely persuasively that the contaminant could be present intermittently and that DNA could remain suspended, and then fall on a particular item.” (p. 67)

“but above all it [the discourse of justification] is based on the erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods tested, the results of which could certainly be called into question…”  (p. 69)

The moment of DNA contamination is often not known
Should a mechanism of contamination have to be demonstrated?  One problem with this argument is that the mechanism of contamination may only be revealed years after the fact or not at all.  In the Jaidyn Leskie case (see below) it was shown that the evidence items were in the same lab closely in time, but the exact moment of contamination was elusive.  Modern forensic DNA testing relies upon the polymerase chain reaction (PCR) technique, which produces many copies from a small number of DNA molecules.  Dr. Donald Riley wrote, “PCR is also very similar to what happens when a clinical infection occurs.  Clinicians have known for many years that a single germ (bacterial cell or virus) contaminating a wound can produce a massive infection.  Similarly, a DNA molecule can contaminate (infect) a PCR and become a significant problem.  The ability of small amounts of DNA to produce false and misleading results is well-known and well-documented within the research community, where the technology originated.  Anyone who has caught a cold from an unknown source, or who has a pollen allergy should have some sense of how easily PCRs are contaminated.  Actually, it is probably easier to contaminate a PCR than to catch a cold since unlike our bodies, PCRs lack immune systems.  The only protection PCRs have is the technique of the analyst, use of control samples to monitor contaminants and careful interpretation.”  Dr. Riley went on to say, “When contamination occurs there is rarely any way to confirm how it happened.”  The argument that contamination must be proved suggests an analogy: that doctors should demand that the patient prove the exact moment at which he or she became infected before the doctor commences treatment. 

In his 2010 report on the Farah Jama case, Former Australian Supreme Court Judge Frank Vincent wrote (p. 24), “Precisely how it [contamination] may have happened cannot be determined as the deposition of the minute quantity of material involved could have occurred in a number of ways.  It is possible to speculate about the probability of transference through various mechanisms, but ultimately pointless to do so.”  Judge Vincent also wrote (p. 45), “Whilst there is no absolute bar to conviction based solely on DNA evidence, the better view is that a conviction should only be returned where there is DNA evidence and at least one other item of evidence present which is consistent with the guilt of the offender.”  The tiny amounts of DNA involved and the ease with which DNA transfers make it very difficult to pinpoint exactly when contamination occurs.  Attorney General Rob Hulls said, “Mr Jama’s case, I think, highlights the need for everyone in the criminal justice system to better understand the nature and the limitations of DNA evidence.  The perceived value of DNA evidence means that extreme care must be taken at every stage of the process: from collection of DNA to the handling of DNA, the testing of the materials and indeed, the interpretation of the results, to the way in which the evidence is presented to juries in criminal trials.”

Lack of good forensic technique
If the forensic police fail to follow generally accepted guidelines in collecting evidence, should the evidence be accepted?  The CSC wrote, “Prof. Novelli had agreed that there are protocols and recommendations, but added that first of all the operator had to contribute his common sense (ud. 6.9.2011, p. Transcription 59.), otherwise it put in question all the DNA analysis done from 1986 onwards.  Does the CSC really believe that poor technique is insufficient ground for considering the evidence to be unreliable?  The laws governing forensic evidence must set the correct incentives in any criminal justice system.  If evidence collection procedures are flawed yet the evidence is accepted anyway, there is no impetus to collect it properly:  The same types of errors may convict innocent persons in other cases.  In the Busco case, Professor Novelli sounded a more cautious note than in the present one:  According to Professor Giuseppe Novelli, ordinarius of genetics at Tor Vergata, ‘There were contaminations among items and for [=of?] the analyses on the corsage and on the bra. The chain of custody of the items did not respect national and international standards for the conservation of items.’”  What common sense actually suggests is that the standards were put into place to avoid generating a misleading result and that if one violates a particular protocol there should be a solid, clearly defined reason.  The CSC provided none; on the contrary, its words are a smokescreen rather than an argument.

The CSC effectively rejected the idea that the onus is on the prosecution to show that the forensic police followed correct protocols.  Not everyone agrees, at the very least.  Based upon a number of cases, a barrister in Australia argued that the prosecution must demonstrate that it has done everything correctly in a preliminary hearing.  Peter Faris wrote, “DNA should never be admitted unless there is corroboration: that is, there must be some other evidence of the identity of the offender.  As for the rest of DNA cases, the solution is to put the burden on the prosecution.”  Mr. Faris continued, “For example, the prosecution would be required to produce evidence to disprove contamination, whether at the scene or in the laboratory.  Strict proof of continuity of exhibits would be required. The prosecution would have to call evidence of the current international statistical procedures. Proof by the defence of international articles and learned writings could be relaxed.”  These are much more sensible guidelines than anything provided by the CSC.

Negative controls and the importance of electronic data files
Another serious objection to the court’s position is its belief that a witness for the prosecution need only testify that controls were run, as opposed to actually producing the negative control data in discovery.  Negative controls are runs in which template DNA is deliberately left out.  A good way to determine whether or not global contamination occurred is to examine the negative controls in the form of electronic data files, because if any DNA shows up, it must be from contamination.  Among the reasons why examination of negative controls should be done using the electronic data files (EDFs), which are the raw data used to construct electropherograms is that this allows the scientist to zoom in on small peaks.  Given the smallness of some of the peaks in the bra clasp profile and of all of the peaks in the knife profile, negative controls in the form of paper copies in which the y-scale were set to two thousand RFUs for example, would be almost useless.  Even if one accepts the dubious notion that the defense bears the burden of proving contamination, then it is indefensible to prevent the defense from having access to any and all documentation that bears on this possibility, including but not limited to having the EDFs:  It is also essential for the defense to examine the laboratory protocols, instrument logs, contamination logs and corrective action files.  Professor William Thompson noted, “Under a guideline issued by the FBI’s DNA Advisory Board in 1998, forensic DNA laboratories are required to “follow procedures for corrective action whenever proficiency testing discrepancies and/or casework errors are detected” and “shall maintain documentation for the corrective action.[19]”  Obviously, the FBI’s rulings are not binding on laboratories in another country.  However, good forensic science, like any science, doesn’t change as one crosses a border from one country into another.  Good forensic principles, such as keeping a corrective action log, deserve to be adopted universally.

Given their importance in detecting contamination, it is not altogether surprising that negative controls themselves may be the subject of forensic fraud.  Professor Thompson continued, “DNA analysts have recently been fired for scientific misconduct, and specifically for falsification of test results, by a number of forensic laboratories, including labs operated by the FBI,[14] Orchid-Cellmark (another large private DNA laboratory),[15] the Office of the Chief Medical Examiner in New York City,[16] and the United States Army.[17]  In all of these cases, the analysts were caught faking the results of control samples designed to detect instances in which cross-contamination of DNA samples has occurred.”  In the case of the fraud committed by Jaqueline Blake, Professor Thompson argued that if her superiors had examined the EDFs, it could have uncovered this misconduct.

The CSC made much of Professor Novelli’s testimony to the effect that he found no evidence of contamination, but what criteria he used were not provided.  The CSC should state whether or not Professor Novelli used the electronic data files in his examination.  If Dr. Novelli did, then he had evidence that has been denied to the defense, which raises the very troubling question of why the prosecution and its witnesses should have access to something that was denied to the defense and its expert witnesses.  If he did not, then his review was incomplete, and it makes his claim that there was no contamination quite dubious at best.  Every DNA expert whom I have consulted or whose thoughts on the matter I have read has been unequivocal in the importance of reviewing the EDFs; that they should be released in routine discovery is a key recurring subject in this blog.  Dr. Mehul Anjaria wrote, "DNA analysts import ‘raw’ data from capillary electrophoresis instruments into software that assists in evaluating the DNA profiles. The DNA analyst can review peaks by zooming in and looking at height, morphology, and location to assist in determining if they should be reported as DNA alleles. The printed data can be edited by the analyst to show only the peaks being reported. Thus, it is imperative that a reviewer have access to the raw data and be able to independently evaluate the raw data in the necessary software to determine if the reported alleles are consistent with the actual data. The laboratory’s interpretation guidelines are another necessity for the review."

Independent case reviews (including but not limited to an examination of the EDFs) turn up problems in about two thirds of all cases, according to Professor Dan Krane.  The Patrick Waring case in Australia is just one of many examples.  Dr. Theodore Kessis commented on the Benjamin LaGuer case:  It is highly improbable that any given forensic DNA laboratory will take it upon itself to contact its accrediting bodies or the press and state for the record how often they make mistakes… To best understand the weaknesses associated with DNA testing we must rely upon the empirical, the occasions in which such deficiencies are revealed either by the press or internal review of a lab’s documentation of such problems by a defense expert. A close look at either reveals that indeed many instances of DNA testing errors have lead to the false conviction of individuals.”

Assertions from witnesses for the prosecution cannot be taken at face value
What should we make of Professor Novelli's claim, which the CSC accepted, of having examined hundreds of pieces of evidence and not observed evidence of contamination?  This is problematic on several grounds.  First, it is unclear what Professor Novelli’s criteria or methods were for proving or disproving contamination.  Therefore, there is a decided lack of transparency in the process.  Second, Professor Novelli did not explain the presence of multiple profiles on the clasp (see part B).  Third, if the CSC accepts the judgment of a prosecution witness over independent experts, it suggests that the court has a strongly pro-prosecution bias.  Nor can it be persuasively argued that Professor Novelli’s credentials in the area of DNA forensics are stronger than those of the independent experts, Conti and Vecchiotti.  Professor Novelli’s main research interest is in the area of medical genetics with some additional research into one specific area of forensic genetics (that of single nucleotide polymorphisms), whereas Conti and Vecchiotti have focused more narrowly on forensic genetics.

Fourth, forensic witnesses make false statements while testifying.  The CSC accepted the prosecution’s claim that negative controls were performed, despite tacitly acknowledging that they were not part of the case file.  In his report on the Leiterman case, Dr. Theodore Kessis highlighted the fact that a negative control electropherogram showed contamination, and that Dr. Milligan labeled it as such.  Despite this, Dr. Milligan later testified that no contamination occurred and that if it had, his reports would have documented it.  In “Tarnish on the Gold Standard” Professor William C. Thompson wrote, “The DNA analysts in the Houston Police Crime lab came up with an easy solution— they simply failed to run extraction blanks (although they claimed in testimony that they had run all necessary controls).” Thus personnel from crime labs sometimes falsely testify that they followed protocols and saw no evidence of contamination.  Furthermore, even if the lab itself detected no problems, independent case review often does (see above).  The implied standard of the CSC (proof by assertion of the forensic police and the prosecution’s witnesses without documentation) flies in the face of the principle that justice must be seen to be done.  It is negligent of the court to accept that when a prosecution witness asserts something as true, the assertion should be sufficient as proof that it is.

Ms. Stefanoni claimed that there had been no contamination in her lab in seven years, but it is difficult to see how a large lab could have an error rate of zero.  Reporting on some crime labs in California, Maura Dolan and Jason Felch wrote, “The number [of errors] reported was small considering overall caseload -- 3,100 over five years – but [UC Irvine Professor William C.] Thompson said mistakes caught by labs ‘undoubtedly’ make up a small fraction of errors. (In fact, he said, labs that report the most are probably better run than those that claim none.)”

The Six-day gap in testing
A central pillar of the CSC’s report (p. 68) is that the six-day gap in testing the knife was sufficient to rule out laboratory contamination:  Dr Stefanoni (technical consultant who wrote the advice art. 360 codaproc.pen.), heard also on appeal, had repeated that there was no evidence of contamination: investigations on the knife had been conducted six days before the last DNA trace of the victim, then the analysis had been blocked for a further six days, a period deemed by the same expert Vecchiotti time to be sufficient to prevent laboratory contamination, as declared in the SAL report, wrongly reported as missing initially.”  Whether this assertion is true is difficult to verify without full discovery taking place.  It is not clear whether all of the DNA electropherograms were released to the defense, and what might be on the possibly unreleased electropherograms obviously cannot be known.  For the sake of argument, however, we will assume that there was a six-day gap.  The CSC cherry picked one statement to bolster its preferred conclusion; both Conti and Vecchiotti still believe that the DNA evidence is unreliable in spite of the gap.  Moreover, the six-day argument does not address the possibility of secondary/tertiary transfer before or during collection of the knife at all (see part B below).

The assertion that a six-day gap is sufficient to exclude laboratory contamination is flawed on at least four grounds.  One, no primary or secondary source in the forensic literature has made this claim, to the best of my knowledge.  Two, any rule of thumb to that effect that was generated with respect to ordinary amounts of DNA may or may not be valid with respect to low template amounts of DNA.  In response to a question of mine Dan Krane replied, “Was Meredith's DNA processed in the lab before the knife?  If so, then I don't accept that six days is sufficient to exclude contamination in the lab.  That would just be bad practice, plain and simple.”  Meredith’s reference profile was indeed generated before the six-day gap and was obviously much higher in amount that the knife profile, which falls into the low template range.  This is doubly problematic.  Generally, one wants to run the reference profile last, because it is in high quantity, but low template quantities of DNA are especially worrisome.  In response to a previous question of mine Dan Krane wrote, “There is absolutely no question but that contamination is a much greater problem in LCN cases than conventional DNA testing. The reasons that it is a greater problem are both because it is easier to detect contaminants ([Sara] Gino's point) and because it is easier to transfer (and to transfer without knowing) smaller amounts of DNA than larger amounts of DNA.”

Three, imagine that a forensic worker uses a tool such as a ruler in his or her examination of one piece of evidence, transferring DNA to it.  Then the ruler is not used again for a week.  If it were not properly cleaned, the ruler would be quite capable of transferring DNA to a new item of evidence.  A recent study of the surfaces and instruments used in autopsies indicates that concerns about contaminated surfaces are well-founded.  The authors wrote, “Using DNA-free swabs, we took samples from instruments used during autopsy and autopsy tables. Surfaces and instruments were routinely cleaned before sampling.  Swabs were subjected to different PCRs to quantify the total amount of DNA and to amplify individual specific STR-markers.  In most samples, alleles that could be linked to bodies that had been autopsied before were found.  Furthermore, we could show that a DNA transfer from the autopsy table to a body was detectable in four out of six cases investigated.”  A review article on trace DNA cautioned, “Fingerprint brushes are able to transfer amounts of DNA between exhibits that could generate profiles and may retain biological evidence for a considerable period of time [205,206].”  Dick Warrington also warned of the dangers of cross-contamination from instruments.  In his report on the Gary Leiterman conviction, Dr. Theodore Kessis wrote (p. 9), “It must be noted however that contamination errors have been documented where no direct processing link between sample and contaminant have been established, raising the specter that a source of contamination can linger in a laboratory for some time.”

Four, in the Jaidyn Leskie case there was a two-day gap in between examination of the condom used in the alleged rape of Ms. P and of Jaidyn Leskie’s clothing at the laboratory.  The police ruled out the possibility that Ms. P was responsible for Jaidyn’s death, and Professor Krane’s examination of the electronic data files suggested that the possibility of a coincidental match was quite unlikely.  Therefore, the only plausible explanation is contamination at the stage of handling evidence.  There was also a one-day gap in the examination of two women in the Farah Jama case, and these two examinations are likely to be the cause of the observed DNA contamination, as discussed in the Vincent Report.  Given that one-day and two-day gaps are demonstrably insufficient to ensure a lack of contamination, then why would a six-day gap be sufficient?

Observation of the collection or testing of the evidence
In the passage from p. 69 (“documented reporting activities carried out under the eyes of the consultants that had nothing to detect…”) quoted above the court seems to put emphasis on the observation of the collection and seemingly also the laboratory work by the consultants employed by the defense.  This raises some serious questions.  With reference to the collection techniques of the forensic police, the court’s stance is nonsensical.  On the one hand, the court notes that the consultants did not object.  Yet the court also wrote, the vehicle of contamination must be identified in order to defuse the data offered by the technical consultants, it not being enough to assume insufficient professionalism of the operators in sampling…” (pp. 68-69)  The court implies that pointing out technical flaws using recordings of the evidence collection long after the fact would not be good enough, despite the fact that this is what consultants or independent experts who are brought into the case later on would have to do.  Equally serious is the fact that no one can see DNA.  Therefore, when one observes the collection of the evidence, all one can comment on is whether or not the police used good technique (a great deal turns on the meaning of the word assumed).  It seems as if poor technique is not sufficient in the court’s eyes unless it somehow demonstrates the vehicle of contamination, which it is quite unlikely to be able to do.  On the one hand, the best technique does not guarantee that contamination will not happen; on the other hand, errors in technique increase the chances that it will. 

If the court also means say that one must raise objections during the observation of testing the items of evidence, then their stance is equally problematic.  Dan Krane observed, “Having the opportunity to witness the testing of samples is of marginal utility at best. Reviews of the underlying data for DNA tests often reveals alternative interpretations of the evidence samples, especially in circumstances were small amounts of DNA are involved and it is difficult to distinguish between signal, noise, and technical artifacts. Observing testing rarely provides any more insights than what should be possible from a review of contemporaneous notes that should be part of a lab's case file. Witnessing testing is far from a cure-all. Problems such as contamination of samples can easily arise before a sample arrives in a laboratory yet could not be detected by an expert observing the testing process itself.” 

Sporadic and global contamination
The CSC wrote, “he [Novelli] excluded absolutely persuasively that the contaminant could be present intermittently and that DNA could remain suspended, and then fall on a particular item.” Despite what the CSC believes, contamination may be sporadic (“intermittent”), as well as global.  Dr. Donald Riley wrote, “Negative controls also can't rule out contamination of individual samples.  The individual samples lack individual signs of contamination if it occurs.”  Other authors whose works are quoted elsewhere in this entry also made the point that contamination can be sporadic.   

Conclusions, Part A
The CSC’s report suggests an almost willful misunderstanding of DNA profiling.  Its failure to put any significant burden on the prosecution and forensic police is out of step with other nations.  The CSC’s stance that contamination must be proved fails to recognize that the exact moment of contamination may never be established even after thorough studies, as were conducted in both the Jaidyn Leskie and Farah Jama cases. A claim that a six-day gap in testing is itself assurance of a lack of contamination is unsupported in the literature and the time gaps in those two cases (during which contamination must have taken place) undercut their contention.  Even if one accepted the dubious argument that contamination must be proved, then it is incumbent upon the prosecution to turn over the electronic data files (including negative controls), machine logs, standard operating procedures, and other relevant forensic data.  Perhaps the most serious error the court made is to accept the assertion that there was no contamination without the methods or standards of proof even being defined.  Under this court’s implicit rules, the prosecution would need only to offer DNA evidence and perhaps testimony from a friendly witness about the lack of contamination to ensure that the DNA evidence would be accepted in any criminal trial.  Even without such testimony, the lack of insistence upon unfettered disclosure would severely hinder the defense from ever successfully challenging forensic DNA evidence on the grounds of contamination or misinterpretation of the data.  Under such rules false convictions based upon faulty DNA evidence are inevitable because a defense based upon a theory of contamination would be virtually impossible to prove.

Part B:  Specifics of this case
Kitchen knife
The CSC ignored innocent DNA transfer to the knife as an explanation for the knife DNA profile.  It is possible that Meredith’s DNA was deposited onto the knife before or during collection by secondary or tertiary transfer, as was suggested by Dr. Alexander Kekule, among others.  Professor Gregory Hampikian’s study of DNA transfer via gloves is further evidence of the plausibility of such mechanisms.  Dried blood is a good source of DNA because small particles can flake off easily, and clothing is a known carrier of DNA.  One can envision tertiary transfer from airborne DNA to officer Gubbiotti’s clothing, to his gloves to the knife.  However, it is not up to the defense to prove a particular route of contamination; instead, it is up to the prosecution to show that they did everything correctly, as discussed in part A.  There was no reason whatsoever for anyone to unpack and repackage the knife.  Compounding the error, it was unwise to assign officer Gubbiotti to anything having to do with evidence collected at Sollecito’s flat, given the fact that he had been to the women’s flat on the same day. Reporting on the Patrick Waring case Estelle Blackburn wrote, “In court, police conceded they had not followed best practice in the case.  Various officers said that the Central Park scene was left unguarded from 1.25am on the night, it was a week before it was searched, and the same officers had visited the homes of the girl and the accused which allowed for contamination of evidence.”  What is not best practice in Perth is not best practice in Perugia. 

The CSC's report does not seriously consider the lack of blood on the kitchen knife the prosecution has argued was one of the murder weapons.  Yet the lack of blood plus the presence of starch call into grave question whether the DNA seen in the profile was actually physically on the knife.  It is doubtful that one can clean a knife of blood and not of DNA, whether the scratch on the knife observed by Stefononi were real or not.  Detergent water should lyse human cells, releasing DNA and other cellular contents.  Bleach is used routinely to destroy unwanted DNA in molecular biology laboratories.  If the knife were cleaned with bleach, the remaining DNA might show evidence of degradation (peaks associated with long DNA fragments would be smaller than those associated with short fragments).  Yet there is no obvious trend of smaller peaks moving from left to right in the knife electropherogram.  Moreover, there is no evidence that the knife was carefully cleaned; on the contrary, the presence of starch suggests that cleaning was not particularly assiduous.  Forensic scientists Elizabeth Johnson and Gregory Hampikian wrote, “it is unlikely that all chemically detectable traces of blood could be removed while retaining sufficient cells to produce a DNA profile consistent with the victim.”

Another fundamental failure of the report stems from the fact that the knife profile is a low template sample.  The CSC wrote,
The discourse of justification, as maintained by the plaintiffs, did not take account of the authoritative voices of dissent concerning the presence of contaminating agents; adequate explanation was not offered as to how this assumption had to cover only some (the most demanding in terms of defense) examined tracks and not others; but above all it is based on the
erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods
tested, the results of which could certainly be called into question, but for their probative value, not for the operations carried out by preceding contradictory technique, from which did not emerge critical profiles at the time, but only in retrospect (about the decision at First Instance had dwelt from p. 289 to p. 298 on an abundance of topics only partially refuted in an appropriate manner, so that equally significant were the observations of Dr Stefanoni, brought to the attention of the court of Second Instance, at the hearing on 6.9.2011).” 

The implicit assertion that the Rome lab was clean enough for low template DNA forensic typing is made dubious by the fact that dedicated facilities have been constructed to carry out this sort of work.  To combat the increased risk of contamination in low template DNA profiling, special facilities and handling techniques are needed, as noted in the New Zealand Herald: “The ESR has spent $1 million building special anti-contamination areas at its premises in Auckland, Wellington and Christchurch. Protocols are being developed for crime scenes where the LCN technique is used and for the handling of samples from collection through to courtroom.  LCN crime scenes will be divided into cold, warm and hot zones hot being the crime zone. Clothes are put on and discarded at each zone to minimise the risk of contamination.”  The crown prosecution service wrote, “The FSS LCN test requires an ultra-clean laboratory and so is more expensive and less widely offered than the standard test.... The site of this bespoke laboratory is remote from other DNA Units, operates stringent entry requirements, is fitted with positive air pressure and specialist lighting and chemical treatments to minimize DNA contamination.”

In the article “Setting Up a PCR Laboratory” Theodore E. Mifflin wrote, “Air handling. For extremely high-performance PCR laboratories that will be involved with detecting very-low-prevalence DNA or RNA molecules (e.g., infectious disease agents in clinical samples), additional measures may be necessary to prevent contamination from the air being recirculated between the pre- and post-PCR laboratories.”  Dr. Mifflin’s main focus is pathology, but his points about low levels of DNA are germane to low template DNA forensics in that both situations use polymerase chain reaction (PCR) to amplify very small quantities of DNA.  With respect to the knife profile it is especially worrisome that Ms. Stefanoni presumably used a Speed Vac to concentrate the DNA.  The sample would have been vulnerable to airborne contamination upon release of the vacuum or to any DNA left in the Speed Vac from previous samples.  At the very least, a control should have been run that was also subjected to the same concentration step.

Indeed, the need for specialized facilities and handling techniques when working in the low template region is recognized even by the prosecution’s own expert witness.  A review article coauthored by Giardini, Spinella, and Novelli stated, “Thus in these conditions [less than 100 picograms of DNA] there is a greater probability of artefacts, partial profiles with fewer alleles, contamination, preferential allele amplification, the complete absence of one allele (allele drop-out) in heterozygous loci and the nonspecific generation of extra alleles (allele drop-in) [78,79]…. Very few laboratories perform low template DNA typing properly, because it requires dedicated facilities and great experience, although there are several published methods for the interpretation of such profiles [80-82].”  At the time of the murder, the Rome lab was not even certified for standard DNA testing.  If a sample is handled in a regular DNA facility, without the special precautions available in dedicated low template DNA facilities, the risk of contamination is unacceptably high.  Even if all such precautions are followed, some prominent DNA scientists find LCN DNA unreliable for forensic profiling.  Nothing in the CSC’s report can be construed even as recognition (let alone an honest assessment) of the special problems inherent in low template DNA forensics or the lack of special precautions taken in the Rome lab.

bra clasp
The CSC’s views on the bra clasp profile are at least equally problematic.  This blog has previously discussed DNA contamination on several occasions.  Van Oorshot and colleagues wrote, “From a theoretical perspective, any DNA deposit that is not immediately relevant to the crime being investigated can be viewed as contamination. In this light, gross or sporadic contamination may appear at any point: (1) before the crime has been committed; (2) in the interval between the crime and securing the crime scene; (3) during the investigation of the scene; and/or (4) within the laboratory.”  There are alleles in the autosomal DNA profile that are unidentified (not belonging to Meredith Kercher, Raffaele Sollecito, or Amanda Knox).  There are also extra alleles in the YSTR profile that do not belong to Raffaele Sollecito.  Even using a conservative threshold of 50 RFU, one locus has DNA from two additional males.  If one uses a threshold closer to that employed in the knife electropherogram, the number of loci with two additional alleles (therefore two additional donors) goes up.  It is unrealistic to posit that all of these alleles were deposited during the crime, leading to the conclusion that it was deposited in a way that is unrelated to the crime.  Therefore, the bra clasp has already been shown to be contaminated by the Van Oorshot definition.

The CSC noted that a cigarette butt was the only other piece of evidence where Sollecito’s DNA was found (p. 68).  They go on to make the fallacious and foolish equivalence that found DNA equals all DNA.  In other words the court believes that the cigarette is the only source of Sollecito’s DNA in the women’s flat.  Yet Mr. Sollecito left fingerprints and presumably used a towel to wash his hands before preparing food.  Unless the forensic police sampled everywhere in the flat, there is no reason to believe that they found all of Mr. Sollecito’s DNA.

They also discount the problem of the six-week gap between the crime and the collection of the clasp, saying that the house was “boarded up.  This is highly misleading:  the police themselves moved Meredith’s mattress and other items from one place to another.  The bra clasp itself moved more than a meter from the point at which it was first seen to the point at which it was collected.  The reason that the forensic police should do their work first is to collect samples before other police officers potentially contaminate the scene.  The American Bar Association’s standards for collecting evidence reads in part, “Standard 2.1 Collecting DNA evidence from a crime scene or other location

(a) Whenever a serious crime appears to have been committed and there is reason to believe that DNA evidence relevant to the crime may be present at the crime scene or other location, that evidence should be collected promptly.”

The reason for being prompt is to minimize the chances of contamination:  “The most important aspect of evidence collection and preservation is protecting the crime scene from the time the first officer or responder arrives until the last piece of evidence has been noted and collected without being contaminated.”

Furthermore, the bra clasp was allowed to decompose during the time that the forensic police stored it.  If it could be retested, it is possible that additional evidence of contamination would surface.  The CSC appears oblivious to this additional handicap that the defense faces, due to the impossibility of retesting.  Some commenters have argued that when a piece of evidence goes missing or decomposes while in police care, the court should be obliged to accept the defense’s interpretation of the evidence.  That principle would produce a more just state of affairs than exists presently.

The CSC wrote (p. 69), “…in a context in which negative controls were made by Dr. Stefanoni, checks that had been stated too superficially to be missing by the experts, simply because they were not attached to the report.”  This is a tacit admission that the negative controls were never turned over to the defense.  An anonymous expert in Italian law (one who is unaffiliated with this case) told me that an Italian lawyer can get anything he wants in discovery.  Yet the electronic data files were never released to the defense during the trial of first instance, nor is there any evidence that they have been released since.  Perhaps negative controls were simply not run (this would not be the first time in the history of DNA profiling that controls were simply left out).  Even if one claims that the negative controls were made available in some format, it is certain that they were not made available in the form of EDFs.

Moreover, the lack of forensic discovery was more pervasive than solely the failure to release the EDFs.  In an article from 25 April 2010 Fiona Ness quoted Greg Hampikian “’It’s fairly routine in the US that I send a request and get what I want. But in the Knox case I haven’t been able to get a copy of the standard operating procedures of the lab and without that, it’s hard to see if they even followed their own guidelines.’”   Not only the EDFs, but also the standard operating procedures and machine logs should have been released but were not.  In addition it is possible that a number of electropherograms were also withheld, some from the same time as the knife electropherogram.

The CSC was silent about the multiple failures of the prosecution to turn over forensic data and about Ms. Stefanoni’s false testimony about the quantity of DNA on the knife.  Coupled with its fecklessness over Stefanoni’s stonewalling with respect to the results with tetramethylbenzidine, the court’s position contradicts any claim to the effect that an Italian lawyer can get what he wants.  The CSC should have insisted upon complete release of all forensic information as a matter of principle, but even more so given the problematic, questionable nature of the DNA and presumptive blood evidence in this case.  Moreover, to demand that the defense prove contamination without simultaneously demanding complete release of all forensic data, especially the negative controls, is absurd:  Lack of complete discovery has repeatedly undermined the defense in this series of trials; therefore, the trials have been grossly unfair.  The Duke lacrosse case might have played out very differently if North Carolina did not have an open discovery law, even one that is not perfect.  Instead of correcting the failures to comply with reasonable discovery requests in the present case, the CSC has put its stamp of approval on them.  If I were an Italian citizen, I would be asking myself whether the CSC would uphold the right to discovery more generally or would it be similarly obsequious to the prosecution.

Forensic technique
The CSC wrote, “So the objective data collected indicating the absence of evidence (already highlighted in the judgment of first instance from p. 281 onwards, which made reference to the video recording of transactions that took place with the precautions of reporting protocols of the forensic team, accustomed to interventions of this nature) giving credit to the hypothesis of contamination…”  They also wrote, “but above all it is based on the erroneous belief that the burden of proof lies on demonstrating the absence of contamination, whereas the demonstration data that emerged from the technical advice was based on properly documented reporting activities carried out under the eyes of the consultants that had nothing to detect, in a clean laboratory environment, activities conducted according to methods tested…” (p. 69) Yet one of the defense consultants, Professor Potenza, did raise objections to the testing.  Therefore, if the court is asserting that no one objected at this stage of the process, it is simply mistaken.

The CSC believes that the evidence collection was handled properly; this is one of the most remarkable and troubling positions it took in the section of the motivations document on the genetic investigations.  For example, how could the CSC know whether or not the lab was clean?  The independent experts, Conti and Vecchiotti, listed some of the recommendations concerning forensic evidence.  “handling of the objects must be reduced to the minimum possible, and the objects must not be reopened [once packaged], not even for interrogation purposes..” and “Once sealed, the containers must not be reopened outside of the laboratory environment.”  This guideline was ignored for the knife taken from Sollecito’s flat, as discussed above.  Conti and Vecchiotti also quote guidelines concerning gloves that will come as no surprise to readers of an entry here, whereas Stefanoni’s beliefs on how frequently gloves are well outside the mainstream.  Alex Heigl wrote, “There was laughter in the courtroom at one point when the jury was shown a video of the detectives collecting DNA evidence, doing the opposite of what the experts [Conti and Vecchiotti] had just described as the correct way.”  Douglas Bremner wrote, “Professor Conti showed a film of the evidence collection that they said they had analyzed frame by frame in Amanda Knox case. ‘There are a number of circumstances that don’t follow protocol or proper procedure’ and the work of the scientific police was not supportable.”  Does the CSC really believe that these errors never happened, or do they just expect a casual observer of this case to take their word for it?

"Everything is possible"
The CSC wrote, “The Court of Second Instance, supported the probable contamination advanced by experts, based on the "anything is possible", which is not an expendable [usable] argument, because of its generality…”  The CSC plucked this phrase spoken under needlessly hostile cross examination and quoted it without context; it is a dismaying and troubling misrepresentation of Conti and Vecchiotti’s position.  The Conti-Vecchiotti report used a variety of sources to document the manifold failures of the forensic team (see above).  What they did not do, and what no one should have expected them to do, was to find a particular moment where contamination must have occurred.

The CSI effect and the cases of Farah Jama and Lukis Anderson
DNA evidence is based upon solid science, but there are some limitations inherent in DNA profiling that deserve due consideration from the justice system.  Despite these limitations DNA evidence can exert an effect on the minds of judges and jurors that is out of proportion to the weight it deserves.  This blog has previously covered the case of Farah Jama on several occasions, but the recent case of Lukis Anderson also underscores the problem of unintended DNA transfer leading to false imprisonment.  A spokesman for the ambulance associated with the paramedics that picked up Mr. Anderson said, “We don’t have any definitive answers as to what may have caused the transmission of DNA going from one person to another.”  The Anderson case is also additional evidence that one cannot easily determine a route of DNA transmission, even when it is obvious that it happened.

Conclusions, part B
The CSC accepted a number of extremely dubious arguments with respect to the DNA evidence in the Knox/Sollecito case while at the same time failing to address good ones.  The CSC did not answer the question of how there could be DNA on a knife without blood.  This paradox alone is strong evidence for contamination.  Remarkably the CSC did not acknowledge the obvious and independently verified failures of the forensic police to follow good practice, both with respect to the knife and the bra clasp.  Its position with respect to the bra clasp, that there was no way for one particular sample of Raffaele’s DNA to have made its way to the clasp, is at best terribly misguided.  The CSC also did not acknowledge that the multiple profiles on the bra clasp force one either to claim many unknown assailants or to acknowledge that it is contaminated.  On top of these other questionable positions, by failing to compel the release of the forensic data, the CSC eviscerated Ms. Knox and Mr. Sollecito’s right to challenge the evidence against them.  The court’s stance not only puts Ms. Knox and Mr. Sollecito at risk of a false conviction, but doing so also greatly increases the odds that future defendants will be convicted on the basis of DNA evidence that is equally erroneous.  One might hope that a nation’s supreme court would be a bastion against the state’s encroachment on an individual’s liberty, but the CSC’s report gave no evidence that such hope is justified; instead, the court has acted as if it has fallen prey to the CSI effect.

Former Australian Supreme Court Justice Vincent summarized the problem perfectly in his report on the Farah Jama case (p. 11).  In other words, the DNA evidence was, like Ozymandias' broken statue in the poem by Shelley, found isolated in a vast desert. And like the inscription on the statue's pedestal, everything around it belied the truth of its assertion. The statue, of course, would be seen by any reasonably perceptive observer, and viewed in its surroundings, as a shattered monument to an arrogance that now mocked itself. By contrast, The DNA evidence appears to have been viewed as possessing an almost mystical infallibility that enabled its surroundings to be disregarded. The outcome was, in the circumstances, patently absurd.”

Monday, August 19, 2013

DNA does fly, and it also transfers quite easily

Part 37 in the Knox/Sollecito case

Update (11/7/2013)
Poy and Van Oorshot wrote, "To further help evaluate the above finding swabs were taken from gloves worn whilst examining a heavily soiled dress during routine casework examination. A significant amount of DNA was retrieved which exhibited a genetic profile that matched that of samples taken from the exhibit."  This is direct evidence that gloves can transfer DNA.

The issues of possible DNA contamination and proper versus improper handing of evidence are at the center of the forensic evidence in the trials of Amanda Knox and Raffaele Sollecito.  This entry will examine two aspects of DNA forensics, dealing with airborne DNA and the need to change gloves when handling a fresh item of evidence.  Both kinds of potential problems need to be recognized and addressed in order to minimize the chances of contamination.

The problem of airborne DNA
Barbie Nadeau wrote, “…the defense claims that the crime scene was badly compromised during the collection of evidence.  Alberto Intini, head of Italy’s national forensic team, disagrees. On the stand, he defended the forensics work and stressed that the crime scene had not been contaminated, especially under cross examination when the defense lawyers tried and failed to prove otherwise. ‘DNA does not fly around like pollen,’ he said…” Ms. Nadeau left her readers with a misimpression.  Later in the trial the defense returned to this issue.  “’DNA does not have wings, but it flies,’ [defense expert witness Sarah] Gino cautioned. ‘In a laboratory where hundreds of samples are examined, the risk of contamination exists and should be taken into consideration.’”

Dr. Intini’s view is seriously in error, as can be ascertained by a number of lines of evidence.  A paper on DNA in fossils (BioTechniques 38:569-575, April 2005) notes, “These molecules are easily spread via aerosol transport. One aerosol droplet can contain many more DNA molecules than one gram of fossil material.” In a discussion of how to collect DNA evidence Dick Warrington advised, “Next, you can prevent contamination by wearing a mask, since you want to avoid coughing and sneezing around the evidence you are processing.”

pipettors and pipet tips
Pipettes are devices used to deliver small volumes of liquids.  They are used frequently when the polymerase chain reaction (PCR) technique is used to amplify tiny amounts of DNA.  For that reason the use of aerosol barrier pipette tips is routine in labs engaging in PCR.  An application note on pipetting explained, “Cross-contamination occurs if improper pipetting causes splashes or drips. Even if pipettors are handled properly, aerosols can be generated that contain DNA molecules and can contaminate the pipettor and subsequent pipet products. This demands increasing efforts to reduce the penetration of contaminants by means of filter tips.”  Gilson, a manufacturer of pipettes, supplies a technical bulletin that reads in part: “For example, if a technician in a crime lab performs PCR on a blood sample, cross-contamination between samples could result in an erroneous incrimination, even if the technician changes pipette tips between samples. A few blood cells could volatilize in the pipette shaft, stick to the plastic of the pipette, and then get ejected into the next test sample. Modern laboratories have taken account of this fact and are devoting tremendous efforts to avoid this problem through the use of filter tips."

laboratory traffic
A number of forensic guidelines are in place to protect against aerosol DNA from one source or another.  For example,  “A ‘one-way traffic’ rule is also observed in the laboratory, once the technician has entered the PCR or the post-PCR rooms, they are not allowed to return to the extraction or pre-PCR rooms until the next day or a complete cloth changing in order to prevent contamination by aerosol particles.”  The problems only get more serious in the low template region of analysis.  Keith Bedford said, “The way I am speaking at the moment, we could probably detect DNA on this pad in front of me.”

laboratory design
Laboratories are constructed in a way to minimize the possibility of contamination due to air flow.  In the article Setting Up a PCR Laboratory.” Theodore E. Mifflin discussed how the design of the laboratory can minimize the chances of contamination: “Air handling. For extremely high-performance PCR laboratories that will be involved with detecting very-low-prevalence DNA or RNA molecules (e.g., infectious disease agents in clinical samples), additional measures may be necessary to prevent contamination from the air being recirculated between the pre- and post-PCR laboratories. In this case, the air handlers need to be separate and the air pressure individually adjusted in each laboratory. In the pre-PCR laboratory, there should be a slight positive pressure compared to the air in the connecting hallway. The post-PCR laboratory, in contrast, should be at slightly reduced pressure to pull air in from the outside and thereby prevent escape of amplicons from the completed PCR samples being analyzed inside the lab (Fig. 2). Finally, the air handlers for the pre- and post-PCR laboratories need to be connected to separate air ducts, and each must lead to a separate location for exhaust.”  Dr. Mifflin’s main focus is pathology, but his points about low levels of DNA are germane to low template DNA forensics in that both situations use PCR to amplify very small quantities of DNA. 

What the police did and did not do with respect to changing gloves
When the forensic police collected evidence at Ms. Kercher’s flat, they did not change their gloves frequently.  One can observe in a series of photographs a failure to change gloves over several minutes and several evidence samples.  Barbie Nadeau reported, “Sollecito’s attorney, Giulia Bongiorno, stopped the crime scene video several times to point out errors. For example, Stefanoni testified that she had changed gloves according to official investigation procedures, but Bongiorno stopped the crime scene video twice to show that Stefanoni’s bracelet and the fold of her glove were exactly the same before and after the time she claimed to have changed gloves.”  Ms. Nadeau recounts the same incident on p. 133 of Angel Face, her book on the murder of Meredith Kercher.

Ms. Stefanoni’s views on when gloves should be changed are found in the English translation of the Massei report.  On p. 203 Massei wrote that Stefanoni "specified" that gloves were changed "every time an object was touched that was particularly soaked with blood, and when it was obvious that the gloves would be soiled;"  Based on pages 204-205, she appears to believe  that the presence of a liquid is necessary to bring about contamination by touch.

What forensic experts say about changing gloves
On page 38 of John Butler's 2005 textbook Forensic DNA Typing, he wrote, "Use clean latex gloves for collecting each item of evidence. Gloves should be changed between handling of different items of evidence."  Dick Warrington is the author of some articles in Forensic Magazine and is employed by a company which makes equipment for crime scene investigations.  He wrote, “If you pick up one piece of evidence and then pick up another piece of evidence you can transfer evidence from the first item to the second item. You can avoid this kind of cross-contamination if you remember to change your gloves before handling each piece of evidence.”  He also advised, “Put on gloves, use gloves, change gloves. Do that every time you touch a piece of evidence. Likewise, use disposable tweezers, scalpels, etc. Change these each time they are used, as well.”  Orchid Cellmark’s guidelines for collecting DNA evidence read in part, “Use clean latex gloves for collecting each item of evidence. It is recommended the gloves be changed between the collection of each item of evidence.”

The need to use fresh gloves when handling a new piece of evidence is utterly noncontroversial.  Yet it is obvious from video of the collection, that the forensic police did not change gloves frequently.  Their failure to follow the consensus view of what is good practice might have contaminated the bra clasp and might have led to mixed DNA samples elsewhere in the flat.  The fact that airborne DNA can compromise an experiment is likewise the consensus view of practitioners of PCR.  Dr. Intini’s ignorance of the dangers of aerosol DNA shake one’s confidence in the ability of a forensic lab under his supervision to combat the dangers of airborne DNA contamination even in routine DNA profiling, let alone low template profiling.

Tuesday, July 30, 2013

An interview with David Balding

Part 36 in the Knox/Sollecito case

Professor David Balding recently published an analysis of the bra clasp DNA.  It may be helpful to explain some terms found in this article.  John Butler (Fundamentals of Forensic DNA Typing) defines the liklihood ratio (LR) as “The ratio of the probabilities of the same event under different hypotheses, and he explains that the prosecution’s hypothesis is usually the numerator, and the defense’s hypothesis is usually the denominator.  A ban is a unit of expressing the weight of evidence (WoE).  This scale is logarithmic; a likelihood ratio of three bans is equal to one thousand.  Some months ago Dr. Balding was kind enough to answer some of my questions about this work.

Does Raffaele Sollecito¹s DNA fall into the category of low template DNA,
and if so, should two separate amplifications have been run?

There's no strict definition of "low-template" but broadly yes the peaks associated with Sollecito are low (but not those associated with Kercher, they are high).  Conti-Vecchiotti discuss a threshold of 50 rfu below which a peak should not be relied on; in the UK, that threshold was used in the past but nowadays as techniques have improved the threshold is often lower, 25 or 30.  However that doesn't matter here as all the peaks associated with Sollecito are well above 50: there is a 65, a 70 and a 98, all the 26 other peaks are above 100.  So it is not extremely low template - many low-template cases are successfully prosecuted in the UK even when some peaks fall below the threshold and so are discounted.  In this case all the peaks associated with Sollecito seem clear and distinct  so I think there can be no concern about the quality of the result as far as it concerns him or Kercher.

Replication is generally a good thing and is nowadays done in most cases in my experience, but not all - one problem is that replication splits the sample and so can increase the chance of not getting a usable result.  But although replication is desirable it is not essential.  (In a sense there already is replication, because each of the 15 loci is an independent test.)  This is all a matter of weight of evidence, which Conti-Vecchiotti paid no attention to: if you measure the weight of evidence properly, that accounts for the extra assurance that comes from replication and gives a stronger result (or conversely gives a weaker result if there is not replication).  But because Sollecito is fully represented in the stain at 15 loci (we still only use 10 in the UK, so 15 is a lot), the evidence against him is strong even allowing for the additional uncertainty due to non-replication.

Are there contributors other than Raffaele Sollecito and Meredith Kercher to the autosomal profiles?  If so, how does the presence of this additional DNA affect the bra clasp as evidence?

Yes, Conti-Vecchiotti identified a further 12 above-threshold peaks at alleles that could not have come from Sollecito or Kercher.  They correctly criticised the scientific police for ignoring these: many do appear to be stutter peaks which are usually ignored, but 4 are not and definitely indicate DNA from another individual.  The extra peaks are all low, so the extra individuals contributed very little DNA.  That kind of extraneous DNA is routine in low-template work: our environment is covered with DNA from breath and touch, including a lot of fragmentary DNA from degraded cells that can show up in low-template analyses.  There is virtually no crime sample that doesn't have some environmental DNA on it, from individuals not directly involved in the crime.  This does create additional uncertainty in the analysis because of the extra ambiguity about the true profile of the contributor of interest, but as long as it is correctly allowed for in the analysis there is no problem - it is completely routine.

Are there contributors to the Y-STR profile other than Raffaele Sollecito?  If so, how does the presence of this DNA affect our interpretation of the bra clasp as evidence?

I haven't looked closely at the Y evidence - there seems no need for it because the autosomal evidence is overwhelming for the presence of DNA from Sollecito.  But from a look at Conti-Vecchiotti, it seems to back up the conclusion from the autosomal profiles: Sollecito's alleles are all represented and these generate the highest peaks, but there are some low peaks not attributable to him; so at least one of the additional contributors of low-level DNA to the sample was male.

The bra clasp was collected about 47 days after the murder, and it was found in a different location from where it was initially observed.  In the interim many people entered the cottage and items from her room were removed.  Are these concerns sufficient for the clasp to be excluded as

The only worry would be if somehow DNA from Sollecito was brought into the room and deposited on item 165B.  I don't know enough about what happened to say if that was likely but I'd guess that people walking in and out of the room etc would be unlikely to do that.

The clasp was collected with gloves that were not clean, not with disposable tweezers (videos here and here).  The glove was handled by more than one person.  Are these concerns sufficient for the clasp to be excluded as evidence? If not, should the clasp be given less weight as evidence because of them?

Same comment - the only concern is if any of this could have transferred DNA from Sollecito onto item 165B.

Would you care to comment on the storage of the clasp after the forensic police tested it?

I know nothing about it.

Did you analyze the electronic data files?  Did you examine the laboratory¹s own protocols and machine logs?

I have only seen the epgs for the autosomal DNA profiles of 165B.  There is an unclear version of them in the Conti-Vecchiotti report, but Prof Vecchiotti kindly provided my with a clean set.

Did you examine the negative controls?


Tuesday, July 9, 2013

The sensitivities of presumptive and confirmatory blood tests

Part 35 in the Knox/Sollecito case


Forensic tests for blood have been the subject of four previous entries, one of which discussed the pseudoperoxidase activity of hemoglobin, one of which discussed the difference between presumptive and confirmatory testing, one of which covered the difference between mixed DNA versus mixed blood, and one of which covered some case histories and which also treated the use of tetramethylbenzidine (TMB) in forensic testing.   Confirmatory tests for blood can be performed by testing for the presence of a biomolecule that is unique to blood:  hemoglobin (the oxygen-carrying protein found within red blood cells), immunoglobulin G (an antibody found in plasma), or glycophorin A, (a membrane protein found on the surface of red blood cells). 

Executive Summary

Positive presumptive blood tests indicate the possibility of blood, but only confirmatory tests allow for the conclusion that blood is present.  Modern confirmatory testing of blood is extremely sensitive, yet was apparently not used on the luminol-positive areas in the present case.  The reported sensitivities of presumptive tests for blood have varied widely. Among the reasons for the differences are differences in concentrations of the chemical reagents, in the times of reaction, whether or not the samples were dried, and whether the reagents were tested under laboratory or field conditions. Luminol is very sensitive, but the intensity of the chemiluminescence decreases with increasing dilution.  Luminol is at best only slightly more sensitive than tetramethylbenzidine (TMB), and any difference is dependent on the particulars of how each test is performed.  The luminol-positive areas in this cases tested negative by TMB; moreover, there was no reported testing of these areas by confirmatory experiments.  Therefore, the luminol-positive substance or substances is (are) more likely to be something other than blood.

A brief overview of confirmatory testing of blood

The National Forensic Science Technology Center wrote, "The line between screening and identification is not always clear. For example, while examining the clothing of a suspect, a forensic biologist might visually locate a brown stain that presumptively tested positive for blood and was then DNA typed. The DNA type is found to match the victim. Knowing that the loci tested are higher primate specific, what conclusions can be drawn?  The only unqualified conclusion that can be offered is that the stain contains DNA that matches the victim. It has not been proven to be blood.  If asked ‘Could the results have arisen because the material tested was the blood of the victim?’ then an answer of ‘Yes’ is justified. However, it would be wrong to report that the material was human blood with a DNA type that matched the victim. The material was not subjected to confirmatory testing for blood or proven to be human in origin."  It must always be borne in mind that the burden of proving the existence of blood falls on the prosecution; it is not the job of the defense to prove that a substance that gives a positive result in a presumptive test is not blood.

Modern confirmatory testing for blood owes its specificity to the use of antibodies, proteins which recognize (bind to) certain molecules (antigens) and which generally do not recognize even closely related molecules.  Modern confirmatory testing owes its sensitivity (see below) to the use of enzymes that are covalently linked (conjugated) to the antibodies.  Such tests are called enzyme-linked immunosorbent assays or ELISAs.  Enzymes are catalysts; therefore, for each antibodt/antigen complex that is formed, perhaps thousands of substrate molecules are converted into products, which may be easily detected because they are often colored.  ELISAs come in a variety of forms that are beyond the scope of the present article. 

The general lack of confirmatory testing in the Knox/Sollecito case

The lack of confirmatory testing greatly weakens greatly the prosecution's conjecture that the luminol-positive areas in the Knox/Sollecito case were the result of blood. A true confirmatory test was apparently never performed on these areas; therefore, one cannot conclude that luminol-positive material was blood.  These areas also returned negative results in the TMB tests.  Yet the forensic police did use confirmatory testing on Rep. 199, which came from Filomena’s room, indicating that they had the ability to perform such tests.  With respect to the luminol-positive, DNA-negative areas in this case, Drs.Virkler and Lednev said, “The prosecution should have used much more convincing evidence to prove the presence of blood.”  Indeed.  Confirmatory tests have become rapid and sensitive; therefore, it is difficult to see why one would not perform them.

The sensitivities of two confirmatory tests for blood

The calculated and reported values of the sensitivies (below) might be too optimistic by a factor of ten, and yet still they would indicate that confirmatory tests for blood are extremely sensitive.  One possible problem is that eventually proteins such as hemoglobin denature, meaning that they lose their biological activity (denaturation implies a change in the three-dimensional shape of a protein or that they undergo chemical alteration).  Denaturation could keep a protein from reacting in confirmatory experiments. That is why the several-month delay in testing Rep. 199 using a confirmatory test was so unfortunate.

Whole blood is about 55% serum and 45% red blood cells (RBCs), although there is some variation in this ratio among individuals. Hemoglobin (Hb) is found in RBCs and immunoglobulin G (IgG) is found in serum.  My calculations of the sensitivities of confirmatory blood tests attempt to normalize to whole blood.

Robert Kerber wrote, "The concentration of hemoglobin molecules in red blood cells is so high (340 mg/mL, 2.3 mM) that they almost could be said to be on the verge of crystallization." (J. Chem. Education Vol. 84 No. 9 September 2007, p. 1541). Therefore, the concentration of hemoglobin is 150 mg/mL in whole blood. 150 mg/mL divided by 2.2 x 10-5 mg/mL = 7 x 106. This calculated dilution factor for detection of hemoglobin is close to the reported values for the HemaTrace test.  One article from the Michigan State Police listed its sensitivity as 0.05 µg/mL, and the authors gave its maximum dilution factor of blood that would still allow for its detection as 1:16,777,216. Another study provides 0.07 µg/mL as the limit of detection.

Williams and coworkers (Forensic Science International Vol. 190, 2009, pp. 91–97) described a sandwich ELISA protocol for immunoglobulin G that have a detection limit of at least 0.1 µg/mL, possibly higher.  ELISA stands for enzyme-linked immunosorbent assay, and it is a type of assay that uses antibodies to bring about specificity.   In the textbook Principles of Biochemistry, Mammalian Biochemistry, seventh edition (McGraw Hill, 1983), Smith et al. indicated that the average concentration of IgG is 12 mg/mL in plasma, therefore its concentration is 6.6 mg/mL in whole blood.  Along with the detection limit of 0.1 µg/mL, this concentration suggests that the test for IgG should detect blood diluted up to a factor of 6.6 x 104, similar to the value of 100,000 reported by Williams and coworkers.

Reports on the sensitivities of presumptive blood tests

The following list is an attempt to survey the forensic literature for information on the abilities of presumptive tests, especially luminol and TMB, to detect diluted blood.  There are wide ranges for the reported sensitivities of each.  A previous blog entry rebutted the suggestion that the reason for the lack of positive TMB results was the supposed greater sensitivity of luminol vs. TMB.  One important conclusion of that entry is that it is commonplace in forensics to follow a fluorescent test such as luminol with a colorimetric test such as TMB.  From p. 258 of the English translation of the Massei report, “She [defense expert witness Sarah Gino] added that, in her own experience, analyses performed with TMB on traces revealed by Luminol give about even results: 50% negative, 50% positive, [276]

L. Garofano, M. Pizzamiglio, A. Marino, A. Brighenti, F. Romani.  "A comparative study of the sensitivity and specifity of luminol and fluorescein on diluted and aged bloodstains and subsequent STRs typing," Int. Congress Ser. 1288 (2006), pp. 657–659.

Kastle-Meyer: 1 part in 10,000
Leucocrystal violet: 1 part in 10,000

Emma Johnston; Carole E. Ames; Kathryn E. Dagnall; John Foster; and Barbara E. Daniel.
J Forensic Sci, May 2008, Vol. 53, No. 3, “Comparison of Presumptive Blood Test Kits Including Hexagon OBTI”

Hemastix 1 part in 50,000 [note: Hemastix is based on the TMB test]
Kastle Meyer 1 part in 10,000

Filippo Barni, Simon W. Lewis, Andrea Berti, Gordon M. Miskelly, Giampietro Lago. Talanta 72 (2007), pp. 896–913, “Forensic application of the luminol reaction as a presumptive test for latent blood detection”

“Luminol can be used to detect the presence of minor, unnoticed or hidden bloodstains diluted down to a level of 1:106 (1 µL of blood in 1 L of solution) [18,63,72].”

Joanne L. Webb, Jonathan I. Creamer, and Terence I. Quickenden.  Luminescence 2006; 21: pp. 214–220, DOI: 10.1002/bio.908, “A comparison of the presumptive luminol test for blood with four non-chemiluminescent forensic techniques”

Hemastix 1:1,000,000 solution
Luminol 1:5,000,000 solution
Both values are said to be consistent with the literature.
“What is most apparent is that the literature available on presumptive blood detection techniques is somewhat variable. There is a great diversity of experimental conditions, which makes comparison between reagents tested by different authors difficult.”

Cox, M. “A Study of the Sensitivity and Specificity of Four Presumptive Tests for Blood,” Journal of Forensic Sciences, JFSCA, Vol. 36, No. 5, Sept. 1991, pp. 1503-1511.

TMB gives a fast positive result at 1/10,000 dilution; a slower reaction is seen up to 1 to 1,000,000 dilution.

Shanan S. Tobe; Nigel Watson; and Niamh Nic Dae´id.
J Forensic Sci, January 2007, Vol. 52, No. 1, doi:10.1111/j.1556-4029.2006.00324.x, “Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular-Weight DNA”

“In the past 50 years, there have been many tests conducted on the sensitivity of presumptive blood tests (4,6–15). The findings of these studies are in great contradiction with each other. Sensitivities for luminol range from 1:200 (11) to 1:100,000,000 (6); from 1:200 (11) to 1:100,000 for leuchomalachite green (LMG) (8); and from 1:2,000 (12,13) to 1:10,000,000 for phenolphthalein (9). The various differences in the sensitivities reported by different researchers of presumptive blood tests are probably caused by differences in reagent concentrations, methods of preparation of samples, reagents and results, and in the type of material containing the blood (4).”

From Table 2
Luminol 1 part in 100,000
Hemastix 1 part in 10,000 within 1 minute; 1 part in 100,000 within 2 minutes

“The luminol reagent reacted instantly, with both the 1:10,000 and 1:100,000 dilution factors producing a blue luminescence. The luminescence lasted for close to a minute. However, both dilution factors were much less intense than the positive control of whole blood. The reaction with the 1:100,000 dilution factor was extremely faint.”

“The Bluestar reagent reacted instantly with the 1:10,000 with a blue luminescent glow but faded within a few seconds. The 1:100,000 dilution showed slight reactivity, with five of the 25 samples showing a very faint positive, which faded in a few seconds. However, both dilution factors were much less intense than the positive control of whole blood.”

Anders Nilsson.  The Swedish National Laboratory of Forensic Science (SKL), Linköping 2006, “The forensic luminol test for blood: unwanted interference and the effect on subsequent analysis”

“Under laboratorial conditions CL [chemiluminescence] was detected from luminol treated stains of the used hemoglobin solution (corresponding to blood) diluted up to 5·106 times. A comparably high sensitivity of the luminol test has been reported in other studies [22].  However the sensitivity is probably not as great under the conditions found at a crime scene and here, depending on several factors, perhaps one may “only” see blood diluted to about 1:10000 [14].”

D. D. Garner; K. M. Cano; R. S. Peimer; and T. E. Yeshion.  Journal of Forensic Sciences, Vol. 21, No. 4, 1976, “An Evaluation of Tetramethylbenzidine as a Presumptive Test for Blood”

“As shown in Table 1, both reagents at the 0.05 M concentration will detect one part
blood in 10 000 parts isotonic saline. Doubling the concentration of the reagent results in
a tenfold increase in sensitivity for both TMB and benzidine. The lowest level of detection of blood by both chemicals was 1 ppm.”

LJ Blum, P Esperanca, S Rocquefelte.  Can. Soc. Forensic Sci. J. Vol. 39. No 3 (2006) pp. 81–100, “A new high-performance reagent and procedure for latent bloodstain detection based on luminol chemiluminescence”

Dilution Light intensity a.u.
1:5                           314,600
1:100                         89,720
1:1000                         6,725
1:10000                        1730
“As expected, the lower the dilution factor, the lower the light intensity (Fig. 7).”

General reference on presumptive and confirmatory tests for blood, semen, and saliva
Kelly Virkler, Igor K. Lednev.  Forensic Science International 188 (2009), pp. 1–17,  “Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene”