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.