A chromatogram is a depiction of what at various times is seen by a detector looking at the end of the column where in this case the liquid comes out and where the liquid itself does not elicit a signal from the detector, but anything that comes out that is not moving liquid, that is not the air stream, that is one of the people, anything that is one of the people walking down the road a mile, comes out, it detects or shows its presence and some measure of how much of it, how many molecules or how high--how large an amount, not concentration, but how large an amount is passing in front of the detector. And depicts that by a peak along the line and every time something comes out there is another peak, if it is seen by the detector.
For the record, 1259-A has D.A. discovery no. 4088 and is a chromatogram on sample Q206. 1259-B is D.A. discovery no. 4094 and is also a chromatogram of Q206.
We are going to show you something on the monitor, doctor. Doctor, could you look at the monitor and compare what is up there with 4088 that you have in your hand, and other than one being a positive and one being a negative do they appear to be the same?
Looks like--looks like it is. What is on the monitor is from that same chromatogram.
Now, that is a chromatogram produced by the FBI on the sock stain, correct, the Q206?
Now, I want to ask you about just a couple of things on this form. I'm going to zoom in a little bit up in this corner, try to here anyway. What does the 160 up in that corner there where the arrow is indicate?
Upper left, I'm sorry. Okay. I see, on the chromatogram itself. It means that what is being monitored by the detecting device is the daughter ion, 160 that has been isolated in the second mass spectrometer and filtered and now filtered and isolated from the other ions and is now impacting on the detector as the liquid is flowing out of the column being transformed into the electrospray, entering the first mass spectrometer and going into the next one where it is converted into a daughter ion so that means that is the ion that is being monitored.
Now, what does that tell you about whether the parent ion--what does that tell you about the 293 parent ion?
Well, that in and of itself only tells me that one of the daughter ions of whatever it was, that went into the second mass spectrometer and which was the 293 daughter ion, broke up and gave off one piece that is a 160.
Now, let me go up a little bit where this peak in the chart is. Can you tell me what the number 37 means?
The no. 37 is the--you see a line on the bottom. What it does, it counts the number of--the scans that go on. This is scan no. 37 that the detector scans the gas as it comes past, and on the 37th pass it has this particular peak.
So that is the retention time of the substance, in terms of number of scans, rather than minutes, but it is the same thing. It is along a movement axis. The liquid flows past and the earliest things are on the right and the current, the latest things, are on the left.
The first of these is the detector took in the signals that it got and it added them all up and it measured how big--how high a peak was generated from it in counting units, and the height of that peak was 24 through 223 counting units. The next line, what it does, it makes measurements of all the points in that peak that it can and gives you an area of that peak, because as you can see, that peak has a height and it has an area. You can see it right on there. And the area is 98,443 counts. In short, the area in counts is four times--takes four times--roughly four times as many counts to cover the area than to cover the height.
Now, does the size of the peak--let me withdraw that. How does the machine know where to draw a triangle? Does that depend on what the operator tells it to do?
Well, it can be that the operator says I want you to count everything that is between a certain space or it can be termed to select that, pick two points that are on a line of where this substance is coming to, because you know, walking past a detector, you don't move past it instantly, nor do all the molecules. At first fewer appear and more and more and more and less and less and less, and so you get this kind of a peak. It is the appearance, the presence and the disappearance that is shown, so you can program how wide a base you want or you can program, draw a line from a point of a certain height. You see on the left the counts are 50, a hundred, up to where the m over Z160. There are these count numbers and so these are height number really in a sense. So it says draw anything that has a height, less than what some of the noise might be, to the next point where you have that same, so you can do it in a variety of ways, but the instrument is instructed over what range to draw the line in the peak.
Now, let me--let me blow up the top of the part of the chart where it says "Mass is 158 to 1--162." What does that tell you?
That means that the eye of the mass spectrometer--the way that it is counting is that it will count anything that comes past it that has a 158 or a 159 or a 160 or a 161 or a 162 m over Z. If it is a high resolution instrument, then it would stop counting 158.1, 158.2, et cetera, but ordinarily it just counts these unit masses, so that anything in that range is seen by it and this way you can actually get peaks in various places. In some places in some retention times there may be some things that have a moderate amount or more of 159 than in others, 160, but that is all within the mass range in which the daughter ion sits right in the middle.
Now, if you set the machinery to just look at the 160 rather than a range from 158 to 162, are you likely to have a higher peak?
Yeah, because it can--I told you, it depends--the number that it gets will depend on how rapidly it is checking off. You know, I see one, I see one, I see one. If it is show than it has got two or three that it sees at the same time, then you get a lower number, a lower number of counts. If you focus just on the 160, then that same counts per minute is counting just 160's. Right now it is counting anything between 158 and 162, and so if they are stuck together, it just counts one count rather than separate counts.
Is it the same kind of thing that you might have if I was taking a picture of you with a video camera, if I was scanning it from the Judge over to the bailiff, back and forth like this, you are going to be on the screen less time than if I just put it right on you?
Right. And so if you needed to sum all those pictures that were taken of me, you would have fewer pictures if it scanned than if you just sat it right on me during that time.
And if you do an even broader scan, the peak would be equivalently less; is that correct?
Now, doctor, could you take a look and tell me if the two charts that are on the screen now are the same as the two that I handed to you, 4088 and 4094?
Yes. They are what appears to be an accurate reproduction of what I have in front of me.
Now, doctor, I have highlighted the peaks on those two charts. Does the chart up on the screen there correlate to the peaks that appear on those two chromatograms?
Now, is the retention time on those two charts consistent with the retention time that the FBI determined you would have with EDTA?
And do both of those charts demonstrate not only the presence of the 293 parent ion, but the 160 daughter ion?
Do you have an opinion as to whether, based on those chromatogram from the FBI, whether the presence of EDTA has been demonstrated on the bloodstain from the sock, Q206?
On the basis of the entire picture, this has been at these concentrations presented as strongly as it can with present technology.
KEY QUOTENo, we don't know how many organic compounds there are in the universe, obviously not, because we are discovering new ones. We are making new ones, but we are also discovering new ones everyday.
Is it possible that there might be some other substance in the universe that gave the same pattern as we see here as EDTA?
Well, of course. You know, there might be anything in the universe. We don't know all the things that are in the universe, so of course there could be.
Do you know of any specific compound, other than EDTA, that has 293 parent ion, the 160 daughter ion, the appropriate retention time and the other characteristics that you described about solubility, other than EDTA?
Well, as far as retention time is concerned, I have not seen any chromatography. This is the first time that it was done under these conditions, so I can't relate it. There is no internal standard, so I can't assign a relative amount to it, but as far as the parent ion, the estimated retention time, that means under the conditions that they use a short retention substance, relatively short retention substance, a parent ion of 293 and a daughter ion of 160, I don't know of any such substance and I have not been able to find any in the search of the Merck index of substances. I have looked and I haven't found any.
Could I have another document marked, let's make it c of that last series because it goes together, which would be 1259-C?
Doctor, take a look at this first and then we will put it on the elmo. Does this appear to be what is called a full daughter ion scan chromatogram for Q206?
Yes. It scans all the masses from 130, which I think is one of the lowest daughter ions of the 293 ion, all the way up to 295, which is just--which is the molecular ion plus three.
Now, I take it what this is, it is looking for the other pieces in addition to the 160?
Doctor, could you look up at the top right-hand corner and tell us what range was being scanned with that chromatogram?
From mass 130, which is one of the break-up pieces of the parent ion, when that breaks up, it gives rise to 130 among others, all the way up to 295, which, as I said, is a piece larger than the molecule.
Now, using my analogy of a television camera, is that--are we talking about a very long range of scan?
So is it fair to say that any particular point that is being looked at is not being looked at very frequently because of the broad range of the scan?
Well, they are not identified by being drawn as triangles or numbered; that's correct.
Do you see any things on there that could be potential indications of the presence of these ions at around the right retention time?
Yes, indeed. We have, within the same range within which we determined before, a countable picture, because it was more focused, remember than in the previous one. We do have the highest peaks in that chromatogram fall into the same retention time range for all of the ions as they did for the ones that were clearly recognized as being 160 ions because of the focus on them.
Now, I'm pointing my pen to the top one, which is the 132 daughter ion. Is that what you are talking about as the peak right in here, (Indicating)? Right in there, (Indicating)?
Yes, except that the way that this--in the electrospray you get movement; you don't get that clear a definition. There are several points in here which you would find with 160 daughter ions.
And now the 160 chart right below that, is that what you are talking about there, that peak is a possible indication of the presence of that?
Now, you will acknowledge, will you not, that these peaks aren't nearly as well-defined as the ones we have looked at before; is that correct?
Yes, of course they are much smaller. The area is smaller and the peak is smaller because all of the energy for counting is spread over the range of 130 to--
--to 295, whereas in the other one it is concentrated. All the energy of counting is to individually count each 160 fragment along with the other ones next--close next to it.
Does this mean that the EDTA that you saw in the earlier two charts has somehow disappeared?
No. You have a less sensitive method, so the same amount of EDTA will show up as a peak that is not as tall and that doesn't have as much of an area. It looks fuzzier. It is looking at it through a somewhat darker glass than you looked at the 160, but you get more information in this run.
Your Honor, for the record, 1260-A has D.A. discovery no. 4084 and is a chromatogram from a testing from Q204, the stain from the back gate, and 1260-B is D.A. discovery no. 4049, is a second test on the same stain from a different day.
Dr. Rieders, let me show you 1260-A and 1260-B. Does this appear to be two chromatograms from two different tests on two different days of the stain from the back gate at Nicole Brown Simpson's condominium?
Well, I can't see the dates. They are different times, but the date is not apparent.
Incidentally, when you were provided with the materials from the FBI the first time, were you able to tell what dates the tests were done on them?
Does this appear to be an accurate depiction of both the chromatograms, 4049, 4084, two tests on the back gate stain on two different days?
Doctor, do the peaks shown on those two chromatograms have a retention time consistent with the presence of EDTA?
Yes, they do. They have the same retention time range. They have a retention time well within the range shown by EDTA.
And do they also demonstrate the presence of both the 293 parent ion and the 160 daughter ion?
Do you have an opinion on whether, based on those chromatograms, there is EDTA present in the stain from the back gate?
In my opinion, yes, it demonstrates that there is EDTA present in that stain.
KEY QUOTENegative controls are samples put through the same procedure as your sample at issue and samples which are known not to contain any added substance that you are looking for. And if run in the appropriate sensitivity they are negative controls because by the method used they will give a negative test result if the--if, a, there is in fact no more or if there isn't any there, and b, that the method that has been applied has worked as well there.
Did Agent Martz of the FBI test an area of the sock that did not have any apparent blood on it?
Well, that means since it is my opinion that the bloodstain contained EDTA, that that came from the blood and not from the sock.
Does that rule out any contribution from laundry detergent or something else that might be throughout the entire sock?
Reasonably certainly, yes, unless of course the blood was dotted with laundry detergent, you know, and nothing else on the sock was.
Now, as to the back gate, did Agent Martz run any negative control on the back gate?
And were those from a swatch taken from an area just away from the blood but had no blood on it?
Well, the interpretation is that you better run four more or do something like that, because you have something there which doesn't make sense.
But the fact that you have three negative controls coming back negative, what does that indicate?
Well, you know, if you want to put it on a majority basis that there is a 75 percent probability that it is truly negative and that the fourth one is something that happened, either contamination, you know, an accident, or you know, something happened to that sample or that it got mixed up. I can't tell you what, obviously, but--
Does the fact that three of the negative controls are negative indicate that there was no EDTA on the control swatch taken from the gate but not from the blood?
Now, were you asked to consider the question of whether the FBI's methods were capable of reliably determining the amount of EDTA that the test detected to any degree of analytical certainty or accuracy?
The amount, no, I really don't think that it is capable of doing a reasonably acceptable quantitative analysis job at all, which is inherent not in what the FBI did, but in the electrospray method.
Were you asked to determine whether the FBI methods were capable of reliably determining concentrations of EDTA in the evidence sample?
No way, and not only because of what I said before, but in order to do concentrations you would really have to know how much sample was in that swatch that you analyzed, how much got wiped off there, how much blood was in there that you have, and there is no way that you can really tell that from the tests that were done.
Was the FBI's testing methodology capable of determining parts per billion, in your opinion?
Now, with the technique that the FBI used we had talked previously about ion count, in other words, how high the peak is. Do you recall that?
With this testing methodology, when they don't use an internal standard, can you relate one peak height to the peak height of a test done on a different day?
Only saying that this is much bigger than that, but which is right, which is wrong or where is right or wrong, you can't say.
Well, does the peak height vary depending on the day that you do the test, or can it?
Well, even with the same day you have as much as a seven-fold variation between two runs each on a 50 parts per million known sample.
KEY QUOTEAnd did you examine--did Agent Martz run several known EDTA stands, 50 parts per million, to see what kind of peak heights he got?
Does that indicate to you as to whether you can use peak height to accurately determine concentration?
Now, let's say if you wanted to use this method as a way of quantifying how much EDTA you found, what would be the appropriate procedure?
You could do it if you used an internal standard that I had mentioned before where the ratio of the area or of the peak heights is constant at a given concentration, and where the two behave in a very, very similar manner.
Now, with the--an internal standard, is that a substance that is very close to the EDTA, but enough difference so that you can tell the difference?
That is the ideal, that you can tell the difference, but that otherwise the behavior is not significantly different. The properties are sufficiently different so you can separate them either with the liquid chromatograph or with a mass spectrometer.
Now, when you would use an internal standard, do you actually put the internal standard with the evidence that you are testing or do you do it in a different run?
No, no, you do it right in the sample. The ideal thing is to--if you have a swatch like he had--is to put the internal standard on the swatch, known amount, and then extract it, really put it on the swatch, let it dry, let it age a while because you didn't just prepare it. You know, it has been around a while, and then extract it. You know how much you should find from that. Now, of course you run also control where you just put neatly the internal standard into the machine and see the amount that I'm going to put on the swatch would show up. If I get a hundred percent of it back at certain height and certain area and then I put it on the swatch, then I put it in the water and so that eventually you end up with a chromatogram like this with two peaks close to each other but far enough apart so that you can tell one from the other.
And does that allow you, since you know how much of the internal standard you put in there, does that allow you to then quantify how much was in the evidence?
Yes, but taking the ratio of, let's say, the height of the known over the height of the unknown or visa versa and comparing that with the standard, because that ratio is going to be reasonably constant.
It is a standard procedure wherever one is available, not only for quantization, but also for retention time characterization. If one moves to a different retention time, so does the other, so again, that relative retention time is kept much tighter that way. And it is almost--it has almost become a requirement of chromatography, in quotes of course.
Now, let's assume hypothetically that the stain from the back gate was put there using blood from a reference tube that had EDTA in it and let's assume also hypothetically that the stain in the sock was placed there using blood with EDTA in it. Were you asked to consider the question of how much EDTA you would expect to find if you then tested those bloodstains eight months later?
Now, is it important, when you are running a sample, if you want to determine how much of something like EDTA is present, that you know how much you start with?
Now, if you had a blood swatch, let's say, from the back gate or a piece of the sock from the sock that appeared to have blood in it, can you tell how much blood is there by just looking at it?
What are the standard procedures used to determine the quantity of blood that might be in a swatch?
Well, you bring it into a water solution, preferably with a little ammonia, because old stains are hard to get out, and you read that in a spectrophotometer for the hemoglobin content or for the hematin content, which is a fairly selective method, or else for the soret band absorbing proteins as you call them.
That is one way. It is a simple way because you don't do anything to the sample. You can handle it afterward, do anything you want to with it. You just put it in a light spectrophotometer and you get the spectrum and at certain wavelength standards you determine how much hemoglobin, which is really what the blood has most of, or if you are dealing with serum or plasma, how much protein based on the soret band which is also an absorption in the ultraviolet region there is.
There are other ways of doing it. I mean, you know more about this probably than I do, because you have been so well-educated in this. You can determine the amount of DNA in it and that tells you how much blood there was, because blood has a fairly known amount of DNA in it. You can also determine other substances in there that are characteristic of blood. For instance, if your background material doesn't have any iron in it, the measurement of iron in a sample like this is a good measure of hemoglobin because most of the iron in blood come from hemoglobin. Again, assuming that this is blood, not serum or plasma. What I usually do is spectrophotometry and/or iron determination and/or copper and zinc, if the--you know, if I have a sample that is diluted blood and I want to know how dilute it is or how much there is, so you can do it, you know.
Well, I don't know what their lab has, but if they have got an incident, I'm sure they couldn't do without the others.
Did you see anything in agent Matheson's work papers indicating that he used any method other than just looking at a stain to determine how much blood he started with from the evidence?
Well, I only saw in his papers here on--he made stain sizes on a--on a cloth and said when I put a drop on I get so much stain and when I put--you know, on another one, I get such an area and the area that I have is--comprises of a fraction of that, which I don't think is a workable way of doing it, unless you do it to exactly the same material, do it many, many times, do averages, standard deviations and other things, so--because what he had wasn't a drop of blood placed on the kind of material he was testing or on anything, for that matter, but in the case of the gate it is something that got wiped off again.
Now, did you evaluate or look at the method that he used to attempt to extract the blood that was in the evidence, the sock stain and the back gate swatch to extract the blood from that?
Essentially he took a--he took what he had, you know, what was available, a portion of it, and he put it into 25 microliters of water.
Let me ask you, hypothetically, if you took, let's say, first of all, an old stain that had been on a swatch for eight months and put it in water, plain water, let's say 25 microliters, and let it soak for 45 minutes and centrifuged it, is that an efficient way to remove all of the blood from the stain?
Objection, improper hypothetical, no facts this evidence, no foundation, your Honor.
It is a way but you won't get a hundred percent, you will get a relatively low recovery from an old stain. You know, a dried stain where it is dried for all that period of time, is bone dry, it won't all dissolve in the water, much less than all, just a small portion or just a small--
What would be an acceptable technique to remove all of the blood from the evidence item?
It is--to remove all the blood really what you would have to do is if you could only use water, you could only use water, is to repeat that. You put it in 25 microliters, you let it sit for an hour, you shake it for an hour, you centrifuge it, you take it out, put it in another 25 microliters and every time you run some kind of a non-destructive test to see are you still getting blood out, are you still getting blood out, until you don't see any more coming out, then you are pretty sure you have gotten it all out. You can even change that by using, for instance, dilute ammonia, which is a better solvent for dried blood than plain water, but it, too, you have to establish that this will all come out by doing sequential extractions and mesh in each one the amount of the sample--blood that is in it in one way or another.
Would you agree that if you were testing an old bloodstain to determine how much EDTA was present and you did not extract all of the blood from the stain, you are going to find a smaller amount of EDTA than might actually be there?
If you don't remove all of the blood from the evidence swatch are you going to--and the blood has EDTA in it, are you going to find less EDTA than is in the whole stain?
Of course. I mean that is very self-evident. If you don't get it all, you have less than if you get it all, whatever it is.
Now, incidentally, are you aware of any other case or any published literature on the issue of determining levels of EDTA in bloodstains in forensic cases?
No, I am not. I don't think there is anything published. I have asked around. I don't know of anyone who has done it.
Would you say that this is brand new ground in terms of that particular test I told you about?
Yes, sure. For EDTA in a blood swatch of this type, it is--it is brand new ground. EDTA has been determined in blood, but not in a blood swatch in a forensic case, et cetera, et cetera, to any published extent or anyway I know.
KEY QUOTENow, the chromatography and the mass spec techniques, those are all techniques that have been around for a long time, haven't they?
Did you see anything in the paperwork that you reviewed indicating that the FBI did any studies to determine the differences between extracting blood from an old stain versus a new stain?
By the way, if you have a swatch that is made from a bloodstain, for instance, on a back gate, can you tell from looking at that swatch how much blood is in it?
A positive control in that context, as in other contexts, is one in which the substance at issue has been added in a known quantity and preferably not only in a known quantity but to a known concentration. That means a situation where you know how much sample you have and how much you are adding. That is concentration. If you don't know how much sample you have and you put it on a microgram, then you know you've put one microgram in, but you don't know what the concentration is because you don't know one microgram per what.
Hypothetically, if you had a bloodstain on a metal gate that was not collected, in other words, was out in the outside environment subject to the weather and other environmental factors for a period of from a day to two or three weeks, what effect would those conditions have on the presence of EDTA in the blood?
You put a wet stain on and let it sit there dry and be exposed for a day or more?
With EDTA blood? There would be some degradation from environment factors. How much should really be determined experimentally, but I'm reasonably sure there would be because it has been done.
Have you reviewed literature on a study that is called the photodegradation of EDTA?
Well, basically that article tells us that if you have EDTA solution, in this case the EDTA in the German river called the Neckar, N-E-C-K-A-R. And if you sample that with EDTA in it on a sunny day in Germany, that the amount of EDTA that is in that solution is going to break down half--half of it is going to break down in less than ten minutes from the sunshine itself. So that is photodegradation. It is because of the energy of the sun rays interacting with the EDTA in the water. The material that was used for that was iron EDTA--was an iron chelate of EDTA. Degradation of EDTA is described for sludge, it is described in the literature for bacterial degradation, so-called biotic, by living organism, and a biotic, by the presence of oxygen and absence of oxygen, so it is known it degrades. If you take a sample of EDTA in blood and freeze it, it will stay there.
If you were trying to determine how much EDTA you would have in an EDTA bloodstain that had been subjected to one day to two or three weeks outside environment on a particular type of metal fence, how would you determine that?
Well, I have done things like this, and in this case it is obvious, you get a material which is as alike as possible to the metal fence, maybe a piece of the metal fence, and you put ten dots of EDTA blood on it and you put on it ten drops--ten dots of blood in other places, of blood that doesn't have any EDTA in it, that is not in an EDTA tube, but it is normal human blood. Normally it contains virtually no EDTA. Then what you do is you analyze the control or more than one control. That means you take a little bit of an area where there is no blood and you take that and try to know how much you have and analyze it. Then you wash off one of the spots that has EDTA and another spot that doesn't have EDTA, you analyze all three and you repeat that everyday for a week or something like that, for a portion of the time. If it just for a few hours that you are interested in, then you repeat it during a period of several hours. You analyze it and you see whether it is broken down, you know, whether the one that was put there an hour ago has less in it than the one that you took off right away and analyzed or the one that you took--was there for a week. And if during that whole period there is no breakdown, then you say, well, I don't know whether it would break down in eight months like you are--or eight weeks or whatever, but at least I know that during that period it is stable. Usually in a substance, my experience is, that a substance that does break down, within a week you will be able to tell that it does. Then carefully can extrapolate say that in eight months there is going to be nothing there or I think there may be some there, but that becomes somewhat speculative.
Now, is that what you have described as a possible method to determine that, is that part of what should be a validation study on the method?
Did you see anything in the paperwork from the FBI indicating that they did any test at all to determine how much EDTA would have been lost under the conditions that we described?
Now, let me ask you about the sock. Let's assume hypothetically that EDTA blood was put on the sock at some point in time and let's assume further than between the time that blood was put on there and the time it was analyzed by the FBI it was examined several times using high intensity lights involving physical manipulation in the process of examining it. Can you tell us whether or not that would result in the degradation of the EDTA that was originally in that blood?
And the one study that you have cited looked at photodegradation and found that it can occur with EDTA, correct?
I understand that, but the one article you have talks about the water quality in the Neckar Valley River.
A delightful place. That is why the Germans call it the romantic highway, but it doesn't tell me a lot about this case.
KEY QUOTEOn the basis of the entire picture, this has been at these concentrations presented as strongly as it can with present technology. As EDTA.
In my opinion, yes, it demonstrates that there is EDTA present in that stain.
For EDTA in a blood swatch of this type, it is — it is brand new ground. EDTA has been determined in blood, but not in a blood swatch in a forensic case, et cetera, et cetera, to any published extent or anyway I know.
A delightful place. That is why the Germans call it the romantic highway, but it doesn't tell me a lot about this case.
Well, even with the same day you have as much as a seven-fold variation between two runs each on a 50 parts per million known sample.