Dr. Gerdes, just going back for a moment, if I can, you described this morning how as far as your paternity testing in your lab that you use databases to calculate some numbers to describe how rare a match is between an alleged father, let's say, and a child, correct?
Now, I would like to shift your attention, if I could, to polymarkers. We discussed a little bit earlier that a polymarker consists of more than one genetic marker; is that right?
As far as this polymarker is concerned, is it also a test that is used using a kit?
And does Roche in fact manufacture, or through Perkin Elmer, market the particular kit that is used to type the polymarkers?
As far as your own personal experience have you used that particular polymarker kit?
We haven't used it for any case work, but I have had an occasion to run one kit's worth which is a hundred samples.
So you have used one kit and you have run a hundred samples through the polymarkers?
I'm not even sure we ran all of them, but we have run at least half that amount I would say.
You haven't done any other testing using any other techniques for these five genetic markers, have you?
As far as presentations, and this would be lectures, have you attended any lectures or presentations reporting information about validating the use of the polymarker in forensics?
This particular set of five genetic markers, are they all amplified at the same time using this thermalcycler, the machine that actually copies the DNA over and over again?
Is there anything unique about that to forensics as far as multiplying more than one marker at the same time?
So in other words, this amplifying a number of markers at the same time is also used in disease diagnosis, correct?
As far as these markers themselves, are they simply five more genetic markers that are used to type samples in a forensic case?
And then in that same vein I would like to turn your attention to D1S80. Have you ever tested that particular marker in your laboratory?
Have you bought or used the kit--well, let me rephrase. As far as this particular marker, is it also tested in kit form commonly?
Is that again simply another marker that is available for use in terms of being another particular genetic marker in addition to DQ-Alpha and the five polymarkers?
Umm, that particular marker is a little different. It doesn't use the dot-blot detection methodology. It is a little different detection and that is the only major difference.
So in other words, the way that the marker is actually typed is a little bit different than DQ-Alpha and the polymarkers, right?
Is it actually read--and you may have read testimony and descriptions of D1S80 results. Is it actually read a little more like the RFLP method?
And there is nothing wrong with utilizing that particular method of telling different types instead using, let's say, a dot-blotting technique?
As far as typing D1S80 by this method, that is more like RFLP typing, is there anything unreliable about that technique?
All of these are--again, the point is that they are all PCR-based tests, they are all susceptible to the dangers because of the sensitivity of the test.
Well, just referring you now to the way that it is typed, setting aside your concerns that you've expressed, is there anything different about typing it by the more RFLP type banding pattern method versus simply dot-blotting it?
Well, it is different. There is nothing wrong with it, if that is what you are--as far as that type of detection system.
Have you ever--and you have described--let me ask one question first. You've described the fact that in your laboratory you use was it FDA-approved tests?
Okay. Is there anything about FDA approval that means you either can or can't use it in a clinical setting?
The regulation is in terms of being able to market a kit for the purpose of clinical testing. The FDA--a clinical lab can run a test that is not FDA approved, they have to just do their own validation and satisfy CLIA that that has been done.
Do you do any testing in your laboratory using techniques that are not FDA approved?
And this is a test that you use in case work with samples that come into the lab to determine whether the CMV virus is present at sufficient levels that some medical danger may exist?
Are there any other tests that you use in your laboratory that are not FDA approved?
I may--I don't believe I have asked. As far as the CMV testing itself, what percentage roughly of the case work in your laboratory does it constitute?
No. The point behind FDA approval is it is generally considered as the ultimate validation for a clinical-based test. That doesn't mean that testing can't be done just because it isn't FDA approved.
And as far as the forensic tests used in this case, they don't have FDA approval, correct?
The absence of that FDA approval does not mean that the tests are unreliable simply because they don't have FDA approval, correct?
Is it correct, Dr. Gerdes, that one general measure of scientific acceptability of a test is the number of labs who are using the technology and cross-checking one another?
Is it also correct that another measure of scientific acceptability of a test is the number of publications that have stated in fact that the test is reliable and it can be reproduced?
Are you aware of any publications that demonstrate that if properly used the DQ-Alpha typing kit produces proper, accurate and reliable results on forensic samples?
As I mentioned earlier, there are numerous publications that deal with and discuss the contamination issues, the sensitivity issues, that we've gone over a number of times here. None of them will have--have come straight out and said that you shouldn't use the kit.
Are there any publications demonstrating the scientific unacceptability of using the polymarker kit in forensic samples?
Are there any publications demonstrating the fact that use of the D1S80 typing kit produces unreliable and inaccurate results?
Again, there are numerous publications that discuss the problems. None of them come right out and say you shouldn't use the kit.
I would like to talk with you a moment about peer review. You are familiar with the term "Peer review"?
It is a process where if you are going to publish an article, the article is sent to a number of reviewers who are your peers, that is, people who have an equivalent training, and they will evaluate that, criticize it, make suggestions, and before the article can be published it has to be approved by a majority of those peers.
The peer review process is an important one as far as scientific literature, correct?
Is it in fact one of the ways the scientific community validates a technique to determine its accuracy and reliability?
Not necessarily. I mean, you are not validating the technique. You are just basically cross-checking with a number of different--a number of your peers to see if your statements are defensible scientifically.
Doesn't this publication process allow other scientists to look at the findings of scientists who publish this material to be able to evaluate whether or not a technique in fact should be used?
They--they--the publication usually is not strictly being offered for the purpose of a flat statement that a particular thing can be used. I mean, it is generally a scientific documentation of observations.
And the peers will look at these observations and see if they have been analyzed in a scientific manner and whether the conclusions are reasonable.
You mention the fact that there were publications that talk about contamination involving PCR, correct?
Those publications serve as warnings to individuals who perform the typing process so that they can incorporate what that publication is showing into their own wealth of knowledge; is that correct?
Well, it depends on the publication. That is too general of a statement. There are publications which are just blind trials, if you will, where--on PCR where the kit was sent, and then there was an observation of contamination. It was simply reported that so many labs had contamination.
Henry Erlich is an individual who was at Cetus at the time this technology was being developed at that particular company and has done work in the area of HLA typing.
HLA again, not only DQ-Alpha, but also at least one of the markers typed in your laboratory?
There is a chapter in that book entitled "Applications of PCR to the analysis of biological evidence." Are you familiar with that?
I believe Henry Erlich is one of the authors if you are referring to the same one. Is that what you are referring to?
With regard to that article, in your view that is an article demonstrating the validity of PCR forensic typing using the DQ-Alpha marker?
I think it discusses the--the issues. I don't think it justifies the validity based on that article. It is basically a review article.
Okay. Does it discuss in fact the uses of PCR to type samples using markers including DQ-Alpha?
Are you also familiar with an article entitled "Polymerase chain reaction PCR amplification and human leukocyte antigen HLA DQ-Alpha oligonucleotide typing on biological evidence samples: Casework experience."
And do you rely on that article in terms of any of the opinions that you've offered in this court?
I consider that publication a one-side--one-sided slant on the cases. It discusses a number of cases, several of which I have been involved with, where there were highly contested issues which are not discussed in that article.
Dr. Gerdes, with regard to these publications about DQ-Alpha--and again, there are a large number of them, correct?
Do you rely on any of those publications that in fact demonstrate this reliability of the DQ-Alpha typing process?
I'm familiar with those publications. I think it is a misstatement to say I rely upon them.
I accept their observations of problems and discussions of those in certain instances. I disagree with certain other statements and I disagree with their conclusions in some instances. It is very general. You would have to take them one at a time.
Okay. Are you familiar with a publication entitled "DNA typing from single hairs"?
Well, that particular paper is simply a demonstration paper. It is a paper that the overall bottom line is that it shows that it is possible to obtain a DNA result on a single hair, and that part of the paper I don't disagree with. That certainly is possible. It doesn't discuss the difficulties that there would be in taking that technology and using it on a routine basis in crime scenes.
Are you familiar with a publication entitled "Genetic markers in human bone: I, deoxyribonucleic acid DNA analysis" by Henry Lee and others?
And to your knowledge was that individual, Mr. Herrin, Dr. Herrin, actually at Cellmark diagnostics at one time?
He is--I'm not sure what his exact title is. He is from the FBI and is involved in the DNA--the Department of DNA Testing or whatever they call their department.
With respect to this publication do you reject Dr. Lee and the other author's conclusions about the reliability of DQ-Alpha typing?
Again, I mean that is a demonstration paper that shows that you can use this technique under controlled conditions to identify individuals based on bone. It doesn't discuss the general problems that can occur, the routine application in labs, and it doesn't discuss the aspect of determining whether a specific laboratory may have significant contamination problems which would render those demonstrations of capability invalid.
One more, if I might, Dr. Gerdes. Are you familiar with a publication by the federal Bureau of Investigation entitled "Validation studies on the analysis of the HLA DQ-Alpha locus using the polymerase chain reaction" by Dr. Comey and Dr. Bruce Budowle?
That publication also deals with contamination and its effects on typing using forensic samples?
Again it is a demonstration paper. What they dealt with in that paper in terms of contamination is one experiment where five tubes were opened on a bench top. They scratched their head over the tubes, closed them up and showed there was no contamination. That is a demonstration. Demonstrates that under those limited number of samples they did not get contamination. From that they conclude that contamination is not an issue. That I disagree with.
Yes, and most of them are, as I said, demonstration papers that show under controlled conditions and in specific instances in particular laboratories it is possible to obtain a type.
Your Honor, may I--the last article that Mr. Clarke referred to I believe has an exhibit number. It came up in the testimony of Mr. Sims, and I don't have it right here, but I think it would clarify the record if we could identify it by this number so that we can flag it with the other testimony.
Dr. Gerdes, you referred to these largely as demonstration papers; is that correct?
Have you ever done such a demonstration paper as far as this particular forensic area of typing is concerned?
Now, I would like to shift your attention, if I could, to the polymarkers. I believe we discussed those a few moments ago. As far as that particular set of five genetic markers, there are similar publications, are there not, in the scientific literature?
There are similar demonstration papers. It is a newer system so there are fewer in number.
Are you familiar, for example, with a publication entitled "Evaluation of the amplitype PM DNA test system on forensic case samples"?
With respect to this article also do you reject its findings and conclusions about reliability of the PM typing system?
I believe they did 16 samples there. It is a demonstration paper and it is interesting that in one of those 16 samples they had a chance match.
In your opinion is this a paper then that does not demonstrate the accuracy and reliability of using the polymarker typing kit on forensic case samples?
Well, I think the analogy here that might clarify this whole thing is basically if I--if you have a clinical kit that someone is using, there may have been some publication as far as the use of that kit, but the ultimate test of any kit, of any method, is how it is used in the field. It is just like products. Just because you put a product on the market and someone says that is a good product, it doesn't mean it is a good product until you've had a chance to look at a large number of individuals who use that product, and if it starts to have flaws, those become apparent. And it is a similar thing in forensics or anything else.
What about when many laboratories use the technique and they r each the same conclusions as the authors, does your opinion still hold true under those circumstances?
In my opinion we haven't reached that stage where there has been enough testimony and discussion and in-depth analysis of the specific problem of contamination and how that can create errors in a blinded fashion in the forensic community.
In your view courtroom testimony is important in establishing scientific validity?
In my view that is--that is like the consumer report. It is what--what--how is it working in the field? We have done these demonstration papers that say it is possible. Now we put it out into the field, we put it on the market. How is it working? What is the feedback from the consumer?
Incidentally, when you refer, Dr. Gerdes, to the first polymarker article, the one we just discussed as being seven laboratories, were you speaking of a different publication?
When you referred to this paper by George Herrin the article on evaluation of the PM system, were you mistaking that paper for another as far as the seven laboratories?
Your Honor, I would ask now that these be marked so that we can know which ones they are.
Oh, several, I'm sorry. All right. Are you also familiar with a publication entitled "Consistency and reproducibility of amplitype PM results between seven laboratories: Field trial results"?
And lastly, in this area, are you familiar with "Amplitype PM and HLA DQ-Alpha typing from pap smear, semen smear and postcoital slides" by Renee Roy--Dr. Renee Roy and Dr. Rebecca Reynolds?
All right. To your knowledge, other than the publications that I have just described, are there others demonstrating the reliability of PM typing on forensic samples?
Umm, there are--there are other demonstration papers that--with regard to the use of the polymarker system.
Can you tell us approximately how many laboratories type the polymarkers in forensic casework?
Incidentally, all of the publications that I have shown you or described the title that we discussed, with the exception of the last one that you are unfamiliar with, they are all from peer-reviewed scientific publications, correct?
And actually the last particular publication I showed you was also in a peer review publication; is that correct?
And then lastly, with regard to the D1S80 system, are you familiar with similar publications demonstrating the reliability of that particular marker's use in forensics?
There are similar demonstration papers discussing the capability of that system to produce a type.
Without going through each of them, would your answers then be the same with regard to those publications, that they are in fact demonstration papers only?
In your view they in no manner establish the reliability of D1S80 typing on forensic casework?
They do not, as I mentioned before. My opinion is the product has to be used to an adequate degree and evaluated in a critical manner, in a critical blinded manner, before you can really determine the errors that are possible with this system.
Can you give us an estimate, for instance, how many cases you think DQ-Alpha typing has been used in in forensic casework just in this country?
With--and I believe you described earlier over 100 laboratories use the technique; is that correct?
I don't believe I asked you about D1S80. Are you familiar with approximately how many laboratories use it in casework?
Again, that is a newer system. It would be fewer than the DQ-Alpha, but most of the labs who have set up PCR for one system fairly rapidly add the next one.
As far as all of these markers, DQ-Alpha, polymarker and D1S80, is it correct that there have been many presentations about their use in forensic casework, and I'm referring to meetings, lectures, symposia and so forth?
That is a fairly common event--let me rephrase that if I may. That is a fairly common manner of the scientific community, discussing work, whether in forensics or clinical work, that is, presentations and symposia?
Do those presentations, conferences and symposia play an important role in not only disseminating but also discussing scientific techniques?
With regard to polymarker and D1S80 and DQ-Alpha, have you attended any such meetings?
You have described how the DQ-Alpha marker was the first marker used following PCR amplification in forensics, correct?
I know when the kit was released, approximately, which was in 1990, and I believe that there was a single individual doing typing probably three, four years before that.
So--and this individual happened to be working in the same area where the method was developed.
Is it correct or it is correct, isn't it, that when you add more markers, in other words, from DQ-Alpha adding additional markers, like PM and D1S80, that that adding of more markers allows the analyst to cross-check results on individual samples?
It allows cross-checking as long as you have totally ruled out the possibility of cross-contamination, because in the case of cross-contamination, remember, you can look at as many markers as you want to and the error is caused by that physical cross-transfer and therefore that type of contamination would not be detected.
Like your clinical use where more markers provide you more information, isn't it also true that adding more markers in a forensic context increases the probabilities that a sample came from one person to the exclusion of others?
You have previously--I'm sorry--previously said you would like to see more forensic markers added to DQ-Alpha, correct?
I think it does add to the capability of detecting the other types of contaminants, which are the--the contaminants due to amplification product carry-over, for instance.
In other words, by adding additional markers you increase your chances of detecting contamination?
And you so testified in 1990 about it would be good if more markers were added to DQ-Alpha?
Since that time six additional markers have been added that are routinely used in forensic casework, correct?
Those six markers that were added to DQ-Alpha were all used in this case; is that right?
That's correct. Since that time it has become more abundantly obvious to me that the type of cross-contamination--
As you add more markers, don't you increase the ability to resolve ambiguities or uncertainties about typing results?
You increase the capability of resolving ambiguities due to amplification carry-over, that kind of contamination, yes.
Incidentally, you have described in your laboratory about the use of PCR with some sample and also using other techniques, serologic techniques?
Well, what I'm really directing my question to is are there any samples that you use PCR with that you will type using serologic techniques?
Well, for the specific cases I am thinking of they give you different information, so for instance, if I want to look at a serological marker for cytomegalovirus, that tells me which individual is at higher risk of having this virus become a problem after they receive their transplant. Someone who has antibody to that before they have their transplant is going to be--they have been exposed to that virus in the past, so that gives me information that that is a patient maybe we should follow more closely by PCR to look directly for the virus after the transplant.
Are there any instances in which you use a serologic technique and a PCR technique as a cross-check in any manner?
As far as this use of multiple markers--and you have described the fact that in this case, at least with many samples, seven PCR markers were used, correct?
Is it correct that serologic techniques that are also used on a given sample, that also adds more markers to act as a cross-check, doesn't it?
Well, you are really again looking at something different. You are looking at an antibody which tells you--if we are talking about an infectious agent, that tells you that that person had been exposed in the past. If they are antibody positive--let's take HIV as an example. If they are antibody positive and then you were to do a PCR and you found the presence of the genome of HIV, then that would confirm the antibody test.
You are familiar with the fact that in this case not only was DNA used but also conventional serological techniques; isn't that right?
If conventional serological techniques were used in this case and results were obtained from stains from genetic markers other than the DNA markers, don't those constitute additional cross-checks of results?
No. My function was to look simply at the science involved and the data involved in PCR.
KEY QUOTEDid any member of the Defense ever discuss with you the existence of serological results in this case?
Is that a factor in your opinion would have any effect whatsoever on any of the opinions you've rendered in this case?
No, because my opinions are as to the scientific reliability of the PCR-based testing and the chances of error due to cross-contamination.
Is it your testimony that such cross-contamination would necessarily never be detected by serological techniques?
I think it is highly unlikely because PCR is the most sensitive method possible and those items would have very little amount of material.
Well, you have raised the possibility that some of the Bundy blood drops may have been cross-contaminated by other stains; is that correct?
One of those stains that you've discussed as being possibly cross-contaminated by other material was item no. 52; isn't that correct?
The amount of DNA in that sample is higher than it is in other blood drops at the Bundy scene, correct; 47, 48, 49 and 50?
52 has approximately 25 nanograms, or I'm not sure what the total yield was, but it has more.
You discussed yesterday the fact that the recommended minimum amount to be used in, for instance, the Roche DQ-Alpha kit, adds two nanograms, correct?
Going back to the serological techniques, as a scientist wouldn't you want to know all the scientific results on a given bloodstain if you were looking at a bloodstain case?
It is not my job to look at this entire case. It was my job to evaluate the reliability of PCR-based DNA testing as an independent piece of evidence.
If you were a serological expert and you were asked to review results on bloodstains from a crime scene, would you want to know about DNA results?
It depends upon whether I was an expert or asked to be an expert on whatever area. I would think that that would be crossing areas of expertise.
Dr. Gerdes, aren't all of the results on these bloodstains important to evaluate what happened?
In my view the way a criminal case should be conducted, if there is scientific evidence involved, you should obtain the expert with the most expertise in every specific type of testing and it is up to someone else in the case that is not a scientist to evaluate the meaning of that. It is basically up to the jury to evaluate--as all of those experts come forward and provide their testimony, it is up to the jury to decide how these pieces fit together. I'm only talking about one piece of this puzzle.
Well, I've heard things on TV, but I have no knowledge or access to specific testing.
Once you heard them on TV, did you feel that there was any need whatsoever to find out what they were?
With respect to this area of cross-contamination, is it your testimony that contamination wouldn't be picked up by serological techniques?
It depends on the specimen, but I think if it is a specimen with very small amounts, the PCR technique is more sensitive than most serological techniques.
All right. Dr. Gerdes, you have described various forms of contamination in your view; is that correct?
And by "Carry-over" that is from amplified DNA possibly getting into DNA that is lower in amount and unamplified, correct?
Is it correct that contamination is just as big a problem at your laboratory as it is in forensic laboratories?
I don't believe that is true, because of the nature of the specimens that are handled and the way they are handled in forensic labs.
Yes. There is a number of differences in the way we handle contamination in a clinical lab as well as the nature of the specimens and that entire board that we talked about.
Dr. Gerdes, with respect to your laboratory, do you continue to offer services to clients, despite these instances of contamination that occur?
You are referring to the actual mechanical manner. How do samples get to your laboratory from somewhere else?
How long a period of time can it be from when that sample is removed from a patient to the time your laboratory receives it?
It is recorded in terms of the request form as to when it was drawn and when it is received. It can be up to a day or two.
Well, one of the processes that occurs when a sample arrives in the lab is you--the first thing the technician does is they look at that time, and if it is too long, they will reject the specimen.
Okay. What would be--I'm trying to think in terms of the testing that you do and let's take CMV.
In that particular case the DNA of that virus is relatively stable and in a blood specimen that is stored at ambient temperature I would say somewhere on the order of five or six days we would still see no real decrease in the amount of DNA that we could detect to allow is to find that virus.
And at the five or six-day period that would be unrefrigerated for those five or six days?
Yes, because we've done previous validation studies to show or assure ourselves that that specimen is still adequate at that point, as long as it is sterile, undamaged, labeled.
In other words, as long the DNA hasn't degraded to the point of not being able to obtain a result, then it is okay to use it?
You have to remember, blood that is in a sterile blood specimen like that is really fairly--it is not going to degrade, it is not contaminated with bacteria, and it is a different--difference why you would--eventually it would degrade, but basically the problem is the cells that are in that blood specimen, if for instance if we are going to do HLA typing, we need those within 24 hours because the cells start to die and it is not a degradation process. There are other complications as to why and it is specific to each test as to why that specimen would be appropriate not.
You described yesterday the fact that in your view other--strike that. You described yesterday that a forensic protocol required that reference samples be separate in time as far as their extraction of DNA, twenty to thirty minutes from evidence samples. Do you recall that testimony?
Do you recall also describing that after a reference sample has had DNA extracted from it, that there should be a clean-up with bleach of that area where that is taking place and then evidence samples can be extracted? Do you recall that testimony?
That all--all--the Twgdam guidelines and a variety of protocols from individual labs that I have read specifically state that reference samples should be handled at a separate time and place than the evidence items.
But as far as this procedure of reference samples, cleaning the area with bleach and then extracting DNA from evidence samples, what forensic protocol requires that?
Well, it is amazing. None of them say it that specifically. Those are pretty common sense things, though, if you are a microbiologist.
Someone who knows about the sensitivity of PCR. I think those are common sense things that you would do.
When you testified yesterday that a forensic protocol requires that, then were you mistaken?
You described yesterday that a forensic protocol required those steps. Was that then not the case?
Basically what I was referring to was the fact that forensic protocols, numerous forensic protocols state that you should handle the reference samples at a separate time and place that evidence items or large amounts of DNA separate than small amounts. And I would have to look in detail in the user guide. I believe there--there is a section in there that discusses these kind of things in the user guide in terms of cleaning up.
Dr. Gerdes, isn't it wiser to extract evidence samples first before reference samples?
The main factor you have to keep in mind is to separate them both in time and space. If you are going to be handling them within a fairly close time, it would be advisable to handle the lower concentration specimen before the larger, so in that case, yes.
In other words, it would be better to extract DNA from evidence samples first because of their potential for having low DNA amounts and then reference samples later because they have higher DNA amounts, correct?
As far as the handling, the reference sample was handled first. As far as the extraction, it was in the opposite order where the reference sample was handled last.
Dr. Gerdes, isn't it true that Collin Yamauchi extracted the evidence first before the known samples in this case?
As far as the extraction of DNA by Collin Yamauchi in the serology room that you have described by photograph, he extracted the lower DNA amount samples first, didn't he?
Have you ever conducted any lab--I'm sorry--any testing in your laboratory involving, for instance, amniotic fluid cells?
Well, it is the fluid that surrounds the baby and it is used for diagnosis of prenatal--prenatal diagnosis.
In other words, you are looking to find out if a fetus will suffer from any diseases, correct?
In that process you are attempting to make a clinical diagnosis of whether or not an unborn child will contract, let's say, cystic fibrosis?
We have conducted testing not on the amniotic fluid itself, but we have conducted testing on cells that were grown from that fluid.
When that type of material is removed from the patient, the pregnant mother, doesn't that material contain a mixture of cells from two different people?
And it can contain a mixture of cells from the fetus which is the cellular material you are trying to test, correct?
Yes. And in that particular instance you know who the mother is and so that allows you to eliminate that--that source.
Isn't that similar to a sexual assault victim in a criminal case who has been raped?
Yesterday you described the fact that in theory a single cell, one cell from a person, a human, could be copied using PCR. Do you recall that?
When you stated that in theory, isn't it correct that there are laboratories that do copy single cells outside forensics?
Yes, there are labs who have done that and it is not something that is--it is not very many labs, but there are labs who can do that.
Isn't it true that the problem of contamination depends to a large extent on how small a sample one is trying to amplify?
For instance, in typing a single cell, a scientist would have to be extraordinarily careful about contamination, correct?
And in fact copying one cell would be presumably what, the height of sensitivity to contamination problems that a scientist could have?
Yes, but they are not typing a single cell from a crime scene. They are--they are typing single cells in a controlled laboratory environment where they can dilute that down with sterile material and know what they started with.
It is an important problem for someone trying to copy very small numbers of cells, correct?
At your laboratory your tests using PCR start with different numbers of cells that are present in a sample; is that correct?
The PCR tests that we use we adjust the DNA concentrations to higher concentrations that would generally be used, relatively speaking, in a forensic setting, and the reason we do that is to avoid as much as we can the contamination problem.
So you set--you made a decision at what level of the number of cells in a sample, before you are willing to use PCR to obtain results that you will report in casework?
You have described your laboratory as being capable of detecting as few as one to five cells in a sample, haven't you?
The initial PCR that we did was just for CMV. Initial studies were adjusted to--for us to be capable of detecting one to five copies of that particular virus. Is that what you are referring to?
Well, the gene you are looking for and the virus is not a cell, so if we are looking for a virus, you are looking for that piece of the virus or sequence of the nucleic acid of the virus and that would be defined as the target. The target is the virus and the amount we would be able to detect would be between one and five.
You then utilized a higher number of--number of cells in this CMV testing, correct?
It is not cells. It is adjusted to the higher number of copies detectable with the PCR system of the virus; not a cell.
All right. Did you in fact adjust it to a higher number of copies to use in your testing?
Yes. The initial studies again on the transplant patients where we looked at the CMV, if we use that kind of sensitivity where we found one to five cells, we were finding it frequently in all of the patients and it wasn't giving us any clinically relevant information, so we adjusted the technique--well, we did this study--a four-year study to determine what was the relevant amount that would allow us to identify patients that needed to be treated as opposed to those who had asymptomatic shedding, then we adjusted the amount so that it would be in that clinically relevant range so that the testing would be useful for patients.
As far as forensic testing and in particular this case, are you familiar with the number of cycles used by the Los Angeles Police Department when they go through this amplification process?
All three laboratories use the recommended number of cycles recommended by the manufacturer?
Far as sensitivity levels isn't it the case that with regard or forensic testing that there are certain sensitivity levels of starting DNA, that is, the amount of starting DNA, that are recommended?
Is it also the case that these numbers of cycles play a role in the ability to copy DNA and obtain results?
Well, in the process this Xeroxing of--molecular Xeroxing of a copy of that, it is basically how long you let the Xerox machine run. If you go--each time you double the amount of DNA and you go 32 cycles, then you have a certain level of amplification ability or copying ability. If you go more cycles, what happens is then just like you leave the Xerox machine on longer, you are going to get more copies, and the longer--the more cycles, the more DNA you are going to make within limits. I mean, eventually you get--it runs out of the things it needs, the building blocks it needs to make DNA, and the whole thing stops.
If anyone--if I were to start a DNA laboratory and I programmed that machine to amplify, say, a bloodstain 50 cycles, would that be a mistake?
Depends on what you are trying to do in terms of protocol. Certainly it would be an increased risk of amplifying very small amounts of material and having them become a contaminant.
And in fact 32, that wasn't a number picked on a board, that is, a dart thrown at a board; isn't that right?
And in fact wasn't the no. 32 arrived at by scientific experimentation and demonstration of the appropriate number of cycles to use?
It is the appropriate number of cycles to use for two nanograms of DNA, which is what the user guide recommends as far as a starting amount.
Well, isn't it correct that these methods have been shown to produce accurate and reliable results using less than two nanograms of starting DNA?
I haven't seen any published detailed studies. There are people who claim that and there are phraseology in certain papers where that is claimed, but I think that there are certain dangers that occur when you get down much below--certainly below a half a nanogram.
And the user guide describes using--I'm sorry--using amounts less than two nanograms as producing accurate and reliable results, correct?
I think there is a phrase in there that says something to the effect that it is possible in an experienced laboratory to possibly type less than that. They don't give an exact amount, how much less, but that phrase is in there.
Hasn't the scientific literature demonstrated typeable amounts, that is, amounts producing accurate and reliable results far below two nanograms?
I haven't seen any paper that goes to a detailed study of tremendously smaller amounts.
All right. Your Honor, I would like to shift subjects and I just inquiring on when the Court was going to take a break?
Controls are used in this typing process to tell whether or not the test is working correctly?
All right. Your Honor, I have an eight-and-a-half-by-eleven piece of paper that I would ask to be marked as--
And Dr. Gerdes, can you--I don't know if it is easier for you to use the monitor to your right.
Sure. That's fine. First of all, you have described previously your examination of various materials from the Los Angeles Police Department, correct?
Your Honor, may I just make a suggestion about positioning, because in this fashion the witness' back is to part of the jury.
And in fact you were and have taken the opportunity to review the testing conducted by the Los Angeles Police Department in their DNA laboratory in this case, correct?
And with regard to that testing, you have also described yesterday having reviewed materials from the DNA laboratory unrelated to this case, correct?
Now, your review of the DNA typing documents from the Los Angeles Police Department in this case reveals that department's, in particular, Collin Yamauchi's, use of a number of controls, correct?
Now, on this particular document that is labeled at the top "Los Angeles Police Department PCR DQ-Alpha testing controls" there is a number "1" and the term "Reagent control." Do you see that?
This is a control that basically--that is why it is called a cloth control. A piece of cloth or a swab that is unused is basically run through the process, including all of the reagents, buffers, the fluid and so forth that are used in this process, and then you look at a typing, go through the same typing procedure, and ask the question is there any evidence of DNA on that particular control. So it is a control for contaminants that would be introduced into reagents or things that are going on along that process, all the way from the beginning.
It comes from the fact that you take a piece of cloth that has nothing on it presumably and you run it through the process.
If a reagent control him shows a DNA reaction, for instance, at the ultimate or the last typing phase of the testing process, what does that tell the analyst?
It tells the analyst that at some time, either in handling that cloth or going through all of this process from the very beginning to the end, DNA has been introduced.
That is one control, reagent control or cloth control, as it is also called, correct?
That is a control utilized by the Los Angeles Police Department not only in their casework in this case, but also in all the other typing that they conducted that you've described yesterday?
That generally refers to the DNA that is incorporated as part of the kit, so the company that sells this kit has a positive DNA sample. It is a 1.1, 4 type and it is incorporated in the PCR process just to ensure that the individual who uses the kit can amplify the DNA and that the strips are working appropriately so that that particular control types as a 1.1, 4.
As far as that amplification control, if during this testing process and ultimately at the final end--first of all, are each of those controls actually typed on a strip at the end?
In other words, they are handled as far as amplification, typing and then the analyst reading the results from the dots just like a regular evidence sample; is that right?
Well, not strictly correct. On an evidence sample there is a rule with regard to a C dot and in these types of controls that rule no longer holds, because of the fact that any DNA, anything at all, present on these types of controls, indicates that there is human DNA that shouldn't be there that is invisible, so it indicates contamination regardless of what the C is. So as far as typeable, that needs to be clarified.
Okay. We'll return to the C probe. What you have just described applies to certainly, no. 1, the reagent control, correct?
No. 2, however, what you just said does not apply, correct, because you expect to find the types 1.1 and 4 as well as a C dot reaction, correct?
Well, actually it does apply if you find the evidence of another dot other than the 1.1 and 4 dot. Perhaps you find a 2 dot or a 3 dot, then you know that there is foreign DNA that has been incorporated into that positive control. So any indication of those kind of dots on this control indicates contamination.
What I asked, though, Dr. Gerdes, was as far as the C probe is concerned and a reaction, the C probe should show up on the amplification control positive, no. 2?
Now, let's turn to--while we are on amplification control positive, if that control is a 1.1 and a 4 and all of a sudden there is a 2 dot there, does that signal the analyst to something on that control?
It indicates on that control that there is a human DNA that has been contaminated on that strip. There is a 2 dot on that positive control and it shouldn't be there.
With respect to no. 3, what is labeled "Amplification control negative," what is that?
This is a control that some people call a water control, and it is incorporated at the stage where you put the sample into the thermalcycler, into the machine that copies things, and that is going to control--basically it is designed to pick up amplification carry-over so if you are going--that product carry-over, this is a stage where that would frequently appear. And so this control is really designed to see if that process, that kind of contamination has occurred.
In other words, that is to help the analyst determine whether or not something has invaded the typing process that may affect the results, correct?
This is a known DNA or known specimen. Most laboratories use a blood specimen. They are drawn from individuals who work at the lab, for instance, or known people at least, and then that is run through the entire process of handling and of extracting the DNA and going all the way through until you get a typeable result on that control for whether or not on a random basis using different kind of types you are able to get a typeable result. And it controls for the extraction process, for the amplification process and for the strips themselves to make sure that they are appropriately typing the sample.
Substrate control--substrate means--that means what something is on, so the typical substrate control would be if you collect a specimen in an area, you would take an adjacent area that doesn't appear to have any obvious blood--if it is a bloodstain, any blood, and you would take a swab from that area and then carry that through the entire process. Now, this is a good control because it goes all the way back to or should at least go all the way back to the crime scene, because if you do that at the crime scene and you carry it through in parallel all the way through, it should tell you not only how much DNA was just sort of like the background DNA in that area at the crime scene, but it also tells you whether during the manipulations, assuming everything was done in parallel, whether during the manipulations that human--any foreign human DNA might have been incorporated.
Well, it is an excellent control in terms of determining background level of DNA as long as--in my opinion it should be altered. I mean, basically it doesn't have a stabilizer in there, and I think it could be improved upon, but it is an excellent control.
You have previously described the substrate control as used in forensics as an excellent control, haven't you?
Now, with regard to, and lastly, the C probe, you discussed that a few moments ago. That is a probe, is it not, used to help guide the analyst in interpreting results as revealed by reactions on the strips?
The C probe is the least amount of DNA on the strip. It tells you whether there is adequate DNA to proceed with the typing result. If the C probe is not present, you don't have enough DNA to type. It is--but the probe--this particular C probe fails as a control if you are dealing with mixtures because in the case of a mixture you don't know what proportion one contributor and the second--the proportion of the different contributors. If there are two contributors, one may have a large amount of DNA adequate to light up the C dot and one may have a small amount of DNA which by itself probably would not have lit up the C dot, but gives you faint detectable signals.
All of these controls were used by the Los Angeles Police Department in this case, correct?
And in fact the Department of Justice uses an additional control what is referred to, and I believe you described it yesterday, their QC sample, correct?
Again, it is a demonstration paper. What they dealt with in that paper in terms of contamination is one experiment where five tubes were opened on a bench top. They scratched their head over the tubes, closed them up and showed there was no contamination. That is a demonstration.
There is no forensic protocol that specifically says to do those things.
No, I haven't published in the forensic area.
I consider that publication a one-sided slant on the cases. It discusses a number of cases, several of which I have been involved with, where there were highly contested issues which are not discussed in that article.
My function was to look simply at the science involved and the data involved in PCR.