1.Marc LeBeau — Direct/Cross

MARC LEBEAU: Well, when I started with the FBI Laboratory, I was thoroughly trained in the relevant areas of forensic chemistry and forensic toxicology, as it pertains to the types of examinations that we are typically asked to perform in our laboratory.

I worked for four years before I started with the FBI Laboratory. I worked as a laboratory supervisor at a medical examiner's office in St. Louis, Missouri. Before that I was a chemistry instructor for the University of New Haven. I worked as a laboratory intern for a private toxicology laboratory and I have also worked as a laboratory technician for Monsanto Chemical Company in St. Louis, Missouri.

MARC LEBEAU: Yes, Rohypnol is R-o-h-y-p-n-o-l. Mivacurium, M-i-v-a-c-u-r-i-u-m. And then, additionally, I coauthored an article on another drug that's called Doxacurium, D-o-x-a-c-u-r-i-u-m. And, again, that's using LC/MS/MS techniques.

I recently was an invited guest reviewer for a textbook on the topic of LC/MS and LC/MS/MS techniques. And then I have also coedited a book on drug facilitated sexual assault that involved, within the chapters, the topic of LC/MS and LC/MS/MS techniques.

MARC LEBEAU: Well, one of the many type of cases that the FBI investigates are corruption by public officials. So it's one of the areas we consider to be a very serious accusation for two reasons.

If there's a crooked public official out there, we want to make sure they get off the streets. And, likewise, if an innocent public official is being wrongly accused of something, we want to at least try to set the record straight to ensure the public's trust in that organization or that individual.

MARC LEBEAU: Well, the LC/MS/MS instrument is actually three different instruments that are linked together. The LC stands for liquid chromatograph. And what this does is it allows us to take a mixture of chemicals and separate them into individual components so that they are presented to the mass spec portion, the MS portion, individually.

So a good example of this would be if you had a sack full of marbles, if you will, and you had small marbles, medium size marbles and large marbles. And if you even complicate it more and suggest that the large marbles were both red -- some were red and some were blue.

If you would pass the marbles, simplifying it, through this instrument, it would separate them out so that initially the small marbles would come out, and then maybe a minute later the medium size marbles, and then a minute later maybe the large blue marbles, and then 30 seconds after that, the red large marbles. So it allows those to be separated and then introduced to the next instrument, which is your mass spectrometer.

The mass spectrometer is an instrument that gives you detailed information about the weight of the chemical that it's analyzing, as well as it applies energy to fragment that chemical into many pieces that presents a very consistent fragmentation pattern, that forms what we call a chemical fingerprint, if you will.

It allows us to, then, search against data bases for what chemicals give you that particular fingerprint, and make the unequivocal identification of the actual chemical. By linking two mass spectrometers together, you are able to do very complex and sophisticated experiments that improves the specificity and selectivity of the analytical procedure.

MARC LEBEAU: Analytical chemistry is simply a subset of the overall field of chemistry. And it involves analyzing matter for specific chemical characteristics.

The most simple form of analytical chemistry that we consider is, you are doing one of two things, you are either given a material and asked to figure out what that material is, try to identify it, or at least characterize it chemically; or you might be given a second -- a separate material and asked to identify the presence of a specific chemical in that material. Both of those are different forms of analytical chemistry.

MARC LEBEAU: As far as numbers that I have authored or coauthored, I believe there's about four or five different articles. As far as peer review, which peer review is simply where a scientist sends a manuscript of their research into a professional journal and the editor of that journal then reviews what -- what they are writing about and then goes out to the field to find experts in that particular area, and asks those experts to critique the work that was done by that scientist that submitted the manuscript.

They usually have a number of suggestions that they send back to the editor who passes them on to the original author. And then they respond to those critiques and ultimately a decision is made whether or not it's worthy of being published in that particular scientific publication.

I'm often asked to be a peer reviewer for a number of the professional journals in my field of study. Usually I have one on my desk at every moment, essentially, to peer review.

MARC LEBEAU: Well, as I indicated, in environmental chemistry, an area where they have used this technique to test water, soil, or perhaps agriculture products.

It's also used in food chemistry to analyze different foods for contaminants, for example, or to verify that certain things that are supposed to be in that are there at a particular level.

It is used in the pharmaceutical industry when they are looking for new drugs; drug discovery trials, when they are trying to identify new metabolites from drugs.

Again, with proteinomics, which is studying proteins, it is probably the most widely used instrument for that.

And it's used to test athletes for the use of steroids.

It is used to look for residues of explosives after a suspected bomb has gone off.

Pretty much any organic chemical can be analyzed using this technique.

MARC LEBEAU: Well, whenever we have a new technique that's introduced into the laboratory, a new instrument into the laboratory, we basically verify that it performs at the same level that the manufacturer claims it performs at. And then we do a number of studies in which we shoot standards that we're used to seeing, chemicals we're used to seeing on other instruments, to ensure that it gives the appropriate response.

Then we employ, when it's time to actually use that particular instrument for a procedure that we're going to apply to evidentiary material for a case, we go through a validation study that verifies things such as detection limit. It may verify interferences to that instrument, any matrix affects from the material that we're going to analyze, and a number of other parameters, depending on what type of method we're developing.

MARC LEBEAU: We use a number of controls to verify that the instrument is working properly, as well as that any procedures that we use on that particular instrument are performing as they are expected to perform. For example, before we use the instrument each day, we inject into it a -- what we call a test mix, which is simply a standard of either a group of analytes that we're used to looking at, or in cases in which we're looking for a targeted analyte, looking for a specific chemical, we will shoot just a standard of that chemical daily, to ensure that we're getting a consistent response, day after day on that instrument.

When we're actually running evidentiary material, we introduce negative controls which are simply closely matched to the evidence, the same type of material that we put through the procedure. We know those are supposed to be negative before we start and we make sure they are negative when we finish, in order to accept those results.

Likewise, we run positive controls. These are samples that we know are to be positive, before we start the examination, and we ensure that they are positive when they are finished in order to accept the data that came out of that run.

MARC LEBEAU: It's used in a number of products, commercial products such as shampoos, detergents, where it's trying to take the metals out of the water that make your water hard. It will help remove those metals so you get a better cleaning action with those detergent or shampoos.

It is found in the paper industry, to help in the bleaching process, to make the paper whiter. It's found in agriculture products such as some fertilizers. You see it in foods as preservatives. And, then, of course, we use it in laboratory settings.

MARC LEBEAU: Again, they are in all those different areas, so there are methods that are related to food chemistry that talk about EDTA and the analysis of it in foods, same with water and in soil, in commercial products such as shampoos and detergents; so there are quite a few.

Again, if you do a search on -- in data bases of published articles for methods for EDTA, there are -- I'm trying to -- I believe there are over 20 that deal with just LC/MS techniques. There are over a hundred that have been used that are published methods that are out there in the literature for a variety of techniques.

MARC LEBEAU: Well, the LC/MS/MS is a very advanced instrument. There are simpler forms of the same technique that could be used. For example, the LC is what is known as an HPLC, a high pressure liquid chromatograph. That instrument, attached to a simpler detector than a mass spectrometer, which is called a diode, d-i-o-d-e, array detector, could be used.

Then, you could also use a technique that's called capillary electrophoresis. There are techniques for using gas chromatography. There are techniques for doing what is called nuclear magnetic resonance spectroscopy and --

MARC LEBEAU: Well, again, we get into the different areas. There are publications in the environment. Environmental studies that have been done looking at the analysis of water, soil. There's agriculture products that have been analyzed by EDTA.

The commercial products that have been analyzed for EDTA using a variety of different instrumental techniques, the whole gamut: HPLC, GC, capillary electrophoresis and, then, any of those techniques linked with a mass spectrometer.

MARC LEBEAU: Well, there are a number of different tubes that are available. And they may or may not have preservatives and anticoagulants in them. We can tell what's in a tube simply by looking at the color of the cap on top of that tube.

The red-stoppered tube, for example, has nothing in it. So when blood goes into it, it's simply blood with nothing added to it. A yellow-stoppered tube has citric acid or citrate in it. Gray-stoppered tubes have potassium fluoride as well as usually potassium oxalate. And, then, we also have the lavender or purple-topped tube that has EDTA in it.

Blood is put into those tubes. They are mixed up so that any preservative in them is equally distributed throughout the blood. And the reason that we have these preservatives and anticoagulants present in the tubes is that, because of this, this is what's called the clotting pathway.

We know that when we cut our hands or something that the blood will clot to stop the bleeding, most of the time. And that's, in part, largely due to the presence of calcium throughout this cascade that leads to the actual clotting. What the anticoagulants tend to do in the tube of blood is tie up that calcium to prevent it from participating in this clotting pathway.

So, again, if we go back to the red-stoppered tube that does not have any anticoagulant or preservative in it, what happens after some time of that blood sitting around in the tube, with calcium that's present from our natural diet and just normal metabolic pathways in our body, is the calcium helps make those red blood cells clump together, or clot. And that, of course, makes it very difficult to use that blood sample in a laboratory setting for doing laboratory testing.

So that's why we have tubes like the lavender-top or purple-topped tube containing EDTA. The EDTA structure is listed down on the bottom portion of the screen and simply what EDTA does, as I mentioned earlier, is it binds metals. Takes metals that are present in that blood sample, such as calcium, to stop the clotting, and iron, which is normally present in our diet, and, again, through metabolic processes binds them up so that those metals are no longer available to work as they normally would. And by binding it up, it -- it stops the blood from clotting.

Here we have the EDTA in the red blood cells. Again, the iron is floating freely throughout the blood, initially, as is the calcium. And once those are mixed up, the EDTA grabs on to those metals, calcium and iron, and binds them and forms a metal complex or a chelate, as I mentioned earlier.

But you will notice that there is -- in that tube, there is still EDTA present, what we call the free acid form of EDTA. That's because they put much, much more EDTA in those tubes than what's actually needed for a standard specimen, for it to work as an anticoagulant and preservative. So you will have excess EDTA present.

MARC LEBEAU: Well, initially, we -- we had to develop the method or we had to ensure that the method would work on the instruments that we were employing for this particular technique. Once that was done, we performed four different validation experiments.

One is what we call our detection limit study. It's simply to determine how low of a concentration, of EDTA, we can detect using our method. We did this two ways. The first way was by taking a solution of EDTA in water and making dilutions of it until we reached a point that we could no longer detect EDTA in that aqueous solution.

Then we took a sample of blood not related to this case, but this blood sample had EDTA in it. It was a standard purple-topped tube that had been filled to the standard level, shaken up. And then we took drops, measured amounts of blood, out of that tube. And, again, we measured to the point that we could no longer detect EDTA.

And as it turns out, with this particular technique, the smallest volume of blood that we can measure out is one microliter, one microliter is one 1 millionth of a litre. At that one microliter drop, we were still able to find presence of EDTA, using this technique.

MARC LEBEAU: That's exactly right. So that was the one step of the validation we performed, which is our detection limit study. We also looked for interferences in normal blood samples that would interfere with this particular analysis, to cause confusion when it came time to interpreting the results. And we did this by analyzing 10 different blood samples, that were collected in tubes that contained other preservatives than EDTA; for example a yellow-stoppered tube, or a gray-stoppered tube, or perhaps a red-stoppered tube.

These are blood samples that were just ran -- from random individuals. And, again, we analyzed that, following this procedure and determined that none of those samples, those other blood samples, had interferences in them that would confuse the results.

Another part of that study was to ensure that our internal standard, which is simply a control that we put into every sample that we're going to analyze, to ensure that that didn't cause any interferences with our ability to detect EDTA in the samples.

The third step was what we called a matrix suppression study. And this is something that is very well known with the use of the LC/MS technique, especially when you use what's called electrospray ionization mode, which we did in this case. And simply what that means is that there are -- it's a well-known phenomenon that there are other analytes present in a sample, that can suppress the signal of the analyte that you are interested in.

So what could potentially happen is, it makes it look like there's less there than what is actually there. We evaluated that to ensure that we weren't getting any significant matric -- matrix affect. And at the very worse we got was a 33 percent suppression in matrix -- caused by the matrix, which is fairly insignificant.

And then the final study that we did was -- I can't think what the final study was, I'm drawing a blank. Can I refer to my notes?

MARC LEBEAU: Well, the analysis, your Honor, is simply that we focused our instrument to look for two of the -- two of the products that are on the screen. We looked specifically for the presence of EDTA that was bound to the iron in the blood. And we chose iron over calcium because it is naturally present at about a 10 to 30 times higher amount than is calcium.

And, then, we also looked for the presence of the free acid form of the actual EDTA. Again, that is because there's so much there in an EDTA tube, that's what you should expect to see the most of, unless it's a case of like a poisoning or something, a metal poisoning.

MARC LEBEAU: A standard operating procedure is simply the steps that you take in order to complete the analysis. And it's done to ensure that you -- that it's done consistently time after time. It includes the information, the background information, and all the materials that you need to perform the analysis, much like a recipe in a cookbook. It tells you what you need and, then, the stepwise procedure to actually carry it out.

But it also includes important things like references you relied upon in developing the procedure and includes the limitations of the method or the results of the validation study.

MARC LEBEAU: Not at all. Many of the cases that we receive in our unit, in particular, would normally be worked by a state laboratory. But, for a number of reasons, they are sent to our laboratory; primarily because, either it would be very taxing on that state laboratory to take people off of their normal casework in order to develop a method, validate the method, and then put it into use; or they may -- in other cases, they may not have the expertise or the personnel in order to do that.

So many of our requests that come in are to analyze for unique or new drugs, or to apply a new technique to a particular analyte. And so we're very, very familiar and it's a normal course of business for us to have to develop a method, validate it, and then apply it to a case.

MARC LEBEAU: Yes, Exhibit 436 is a manuscript out of the Journal of Analytical Toxicology published in November/December of 1997, that's entitled The Analysis of EDTA and Dried Bloodstains by Electrospray LC/MS/MS and Ion Chromatography. And this is one of the -- this is the primary article that we relied upon in order to develop the method in our particular -- in this particular case.

The second article, Exhibit 437, is from a journal that's entitled Analytical Chemistry. And the title of the article is Determining EDTA in Blood. And, again, we relied upon this article to help us along the way as we were developing and analyzing specimens in this case.

MARC LEBEAU: All right. Probably the most significant improvement I think we undertook when we developed our method is, we introduced what's called an internal standard as I alluded to earlier. This is simply, for each sample we're adding a positive control to that actual sample.

It's a chemically modified version of the same analyte that we're looking for. It's simply made a little bit heavier than the normal analyte. And it allows us to get a very accurate assessment as to if the actual sample itself worked, not just the batch run of samples, but it allows you to assess each individual sample as to whether or not it should pass or fail your quality assurance protocols and quality control protocols you have set up.

We introduced that, which neither of these papers did. We also -- We looked for the presence of the free acid form of EDTA in one of the techniques that -- that the first reference, Exhibit 436, did not look for. So we added an additional test, if you will, to our protocol, that allowed us to kind of take a three prong approach to looking for EDTA in these samples.

And we used -- Furthermore, we used a different LC/MS/MS instrument than what was used in this -- the 1997 article in the Journal of Analytical Toxicology. The instrument we used is newer than the one they used in this particular procedure. And improvements have been made that I believe helped us eliminate one of the concerns that they reported in this particular paper, and that is of carryover of the samples from one sampling to the next.

MARC LEBEAU: Again, if I can go to this presentation, the idea was, what we were asked to do is determine if someone took a purple-stoppered tube of blood that has EDTA in it, takes the cap off that tube, and then pours a drop, or many drops out, onto the surface, if someone comes along at a later date, swabs up that dried bloodstain, are we able to, then, find, on that swab, from that stain, the presence of EDTA and EDTA linked with iron.

That's the scientific hypothesis, is that we should be able the find the presence of free acid EDTA, as well as the EDTA that's bound to iron, off of that stain that's on the swab, that's collected from a bloodstain.

MARC LEBEAU: Again, we ran negative controls, these were blood samples that were not put into an EDTA tube and then they were -- samples of the blood were applied to swabs. Those swabs were carried through the whole extraction procedure. They were to be negative from the start and at the end of the analysis they were indeed negative.

We ran positive control samples. We did this two ways. We ran a positive control sample of blood from a lab volunteer who, again, their blood sample was put into a purple-stoppered tube that contains EDTA. That blood sample we expected to be positive at the end of the run and it was indeed positive.

But, additionally, we took the blood sample that was provided to us from Steven Avery. We put that on a swab and ran that through as a positive control. It should have had EDTA in it because it was in a purple-topped tube, and it did, indeed, have EDTA in it, served as a positive control.

And, then, as I alluded to, the internal standard that we introduce into every sample, that is another control that allows us to assess that that actual sample worked as expected. So that was a third type of control we used.

MARC LEBEAU: And the idea is, the whole premise behind our protocol that we developed was that we should be able to find EDTA and EDTA bound to iron on that swab. And our validation showed that we can do that. We absolutely can do that.

Then I was asked about the results in this particular case. And the results in this particular case, with the swabs we received, we did not detect EDTA and EDTA with iron linked to it. So this is specifically for the results on our -- on the case at hand today.

MARC LEBEAU: Well, specifically, in this article that we relied upon, The Journal of Analytical Toxicology, they talk about it in relation to old bloodstains. They -- They looked at bloodstains that were two years old and were still able to identify EDTA after two years of storage at room temperature.

Likewise, we performed a similar analysis in our lab in which we looked at bloodstains that had been put onto cards, spot cards, EDTA blood that had been placed on the spot cards in May of 2004 and stored at room temperature up until they were analyzed, just last week. And we were able to identify the presence of EDTA in every single one of those.

MARC LEBEAU: Well, I can explain it two ways. One way is talking about concentration of EDTA. And as I explained earlier, we were able to do decreasingly lower and lower concentrations of EDTA in a water solution.

At that -- Using that technique, we could identify 13 micrograms per milliliter of EDTA. And that's -- it's a number. It doesn't necessarily mean a whole lot unless you are a scientist. But likewise, what we did is we took a tube of EDTA blood and we did spots of that blood, to the lowest volume that we can accurately measure out which is one microliter. And even that one microliter drop of EDTA blood, which is the equivalent of about 1/20 of a drop of blood, even that little amount, we were able to find the presence of EDTA in.

MARC LEBEAU: No. No, the research section of that old CV from three years ago, were different topics of research that I had engaged during my course of study at different universities. So, for example, the last two items, Detection of Drug Facilitated Sexual Assault and GHB in Drug Facilitated Sexual Assault, those were my dissertation topic for my doctorate.

The topic about Trace Elements and Hair, that was an undergraduate research project I did at the Central Missouri State University in Warrensburg, Missouri, was part of a senior level project. The Ceftriaxone in Foul was a graduate research project I was employed upon at my job when I was working at the St. Louis County Medical Examiner's Office. It was a side project that my supervisor asked me to work on with him. And --

MARC LEBEAU: So, your Honor, as part of any publication that employees at the FBI Laboratory put out, we're required to have an additional peer review step conducted by employees, within our organization, to ensure that the science is valid, so we don't embarrass ourselves before it goes out to a peer reviewer. I was an internal peer reviewer on the article that is in the Journal of Analytical Toxicology and I made comments on that article and sent it back to the researchers that were involved in it.

After my comments were addressed, it then went out to the actual journal and that editor of the journal employed some reviewers to look at it. Now, I don't know who those reviewers were and I never saw that review.

MARC LEBEAU: I think that's the message getting a little mixed with an in between messenger. What I told him was, that since we had moved our laboratory in 2003 from Washington D.C. to Quantico, Virginia, we had acquired a number of new instruments and a number of the instruments that we had at the old laboratory did not come with us.

So what that involved is, if we had not used any protocol that moved with us to the new laboratory, we had to essentially revalidate, to some extent, if we were putting it onto a new instrument that it, you know -- the instrument had never been used before, for this particular analysis.

So my message to Mr. Gahn was that we had not used the protocol since the O.J. case and that we would need to bring it up to standard with today's accreditation standards for our accrediting value; we would have to make sure that it met all of our internal quality assurance requirements, because since the O.J. case we have gone through four different quality assurance systems in our laboratory, each one is another step up --

MARC LEBEAU: The time frame wasn't shortened, there were no guarantees that we would get it done, never made a guarantee that we would. But what changed was, simply, we applied ourselves to that and no other case. Myself and some of my staff, this is what we worked on. And we worked long hours, there were weekends involved. There were, you know, more than the standard normal hours of operation.

That's what happened, you know, we decided that we would help out. We committed that we would work out on the case and we would try our best to complete all of the work that was required in order for it to be here during this court proceeding. But, again --

MARC LEBEAU: No, I disagree. There are certainly standards that we have to employ that are based on the FBI Laboratory's quality assurance program.

And those are very stringent protocols and requirements that are, in essence, based upon our accrediting body's requirements. And that, we have actually stepped up to a more stringent accreditation program where we're following what's called the International Standards of Operation or ISO protocols. And --

MARC LEBEAU: Not exactly what -- the conclusion I came to was, we were asked to verify if this chemical, which I will just call SMC, was present in her specimens. And National Medical Services had found this chemical in every specimen they collected from her, from the heart, to the kidney, the liver, the brain, and fat tissue.

We were not able to confirm it in any of them, except for one or two specimens. So we did confirm that this chemical was present in those specimens that were collected from an exhumed body.