Sexual Assault: Obtaining DNA From Evidence Collected up to a Week Later

Lois Tully, Deputy Chief, Investigative and Forensic Sciences Division, Office of Science and Technology, National Institute of Justice, U.S. Department of Justice

Lois Tully: Are there any other DNA dinosaurs in the room here today? So you remember those old days when our DNA methods were much more labor-intensive than they are now. We worked with radioactive labels, and it would take sometimes several weeks to get an X-ray or a radiograph with some results.

But in those days I do remember that the methods we used — they were very powerful statistically. It gives very good statistics when it worked. But in order for the DNA testing to work in those days, you had to have a fair amount of DNA, and it had to be [in] relatively pristine condition. And, as we know, when we collect crime scene evidence, that’s not normally the case. In the last 20-plus years, there have been some great advances. There’s been the polymerase chain reaction and other methods that have made DNA testing more sensitive, more specific.

We’ve found some other DNA markers that have been a great value to us in the laboratory, including DNA markers on the Y chromosome. The Y chromosome, as you know, is found exclusively in males, so especially in sexual assault cases, we now had a new tool that we could use to investigate these cases and analyze crime scene evidence.

Let me just say one thing: I think there’s a few empty seats in the front. They promised me there’d be more room in this room. They took some of the tables out. If there are other empty seats can someone point to them, so we can have everyone sit down.

We’re here today to talk about DNA testing from a variety of perspectives. We’ve done some work over the past few years — funded by NIJ — in the laboratory to help improve the methods used to analyze DNA. Dr. Jack Ballantyne from the University of Central Florida will be speaking about that research.

We’ve been fortunate over the past few years that we have strengthened our partnerships, with not only the social science side of NIJ, but also communities outside of our traditional crime laboratory communities. And we’ve been fortunate to have been working over the past few years with Dr. Pat Speck from the University of Tennessee. She’s a sexual assault nurse examiner, and she’ll be one of our speakers today also. Pat refers to herself as our “token nurse,” but she’s really been much more than that. She’s the person we go to, to get that perspective. You know, we can do some great research in the lab, but it needs to get out into the field, and we need to know what the issues are that will impact the field, so that the research has the highest utility and value. She’s been very helpful to us in giving us that guidance and giving us that perspective.

One of our other panelists is Dr. Mickey Prinz from New York City DNA laboratory, the Office of the Chief Medical Examiner. It’s important to bring in that perspective also, so we can do the great research; we can work with the SANE community — the sexual assault nurse examiner community — on evidence collection. But what are the other impacts of this work? How does it impact the laboratory? In prioritizing cases, in the throughput, et cetera? So Mickey will be presenting to us that perspective.

I’m excited. I think this will be a great panel. And I encourage you at the end, we should have plenty of time for questions; I encourage you to ask questions. And let’s have some discussion. I also need to let you know that this session is being videotaped by CBS News, so that’s why there’s a camera in the back.

With that I think we can get started, and our first speaker is Dr. Jack Ballantyne.

Jack Ballantyne, Professor of Chemistry, University of Central Florida, and Associate Director of Research, National Center for Forensic Science, Orlando, Fla.

Ballantyne: But it’s 30 years. In the course of doing this 20 years operationally and 10 years in the academic setting, I’ve personally or been responsible for the analysis or the interpretation of data or the checking of data for thousands of rape cases. All right. This is typical of people who’ve been in this business for a long period of time. In forensic science we actually deal with more rape cases per person than many of the medical personnel. Now we don’t go into as much detail in some ways, but in terms of the interpretation of data and the analysis of data and what we obtain from evidence from rape cases, we have a lot of knowledge — systemic knowledge and corporate knowledge, so to speak.

However, the knowledge is still lacking, and that’s why we started this project that I’ll talk about. And basically trying to extend or examine the period at which we can still obtain the male genetic profile from the male donor in a rape-type case after sexual intercourse.

So, my own research activity is we’ve spent a lot of time working on Y chromosome markers. I’ll talk about the postcoital time interval, but we’ve also been working on body fluid identification. We’ve realized that in rape cases that positive, good markers for vaginal fluid as well as menstrual blood will also help in the rape case analysis setting. So we’ve taken a multifaceted approach to rape case analysis. But all I’m going to talk to you today, since that’s the topic, is the one of postcoital persistence of the DNA profile — not of sperm and EP, et cetera, but the DNA profile itself.

The problem that we’re faced with is some rape victims provide samples greater than 24 to 36 hours after the incident takes place. As we know — many of you are DNA analysts, probably most — the ability to obtain the standard STR profile of a semen donor diminishes rapidly as that postcoital interval is extended. And that’s based upon the usual personal experience, as well as some studies. There are some studies. But, you know, in forensic science we like to talk over coffee or other types of libation. And it’s over these cups of coffee or other libations that these theories come up. When you stop to analyze a theory, some of them are not based on any actual hard data. That means [for] people like me, there’s an opportunity for improvement then, right? We can do research, which is good. So between 24 and 48 hours, it’s quite difficult to obtain a standard DNA profile. It’s not impossible, but it’s difficult. And greater than 72 hours is normally not possible. I’m not saying it’s impossible; it’s normally not possible.

Now the problem with that is that we have a discrepancy in the literature between the classical serology forensic science studies and reproductive physiology studies. Because you know, you go to court, and how long does sperm persist? Three days, something like that. But there’s variation in the living rape victim. And that’s what we say often or many of us have. I’ve certainly testified to that myself. But the problem is that it’s very clear from the literature that sperm, in fact, persist much longer than that in the female reproductive tract. So, for example, in the cervix, sperm persist in the cervix for seven days and beyond after sexual intercourse. Many, many studies have shown that. So there’s a big discrepancy here now between what — the sperm are present somewhere and that we have an inability to typically obtain a standard profile.

So we thought that this was an interesting problem to look at — not problem, just a study. Before I start, sperm are lost over time after intercourse takes place. You lose sperm due to lavage, drainage, you have degradative changes — the normal metabolic changes that take place that basically destroy the sperm over a period of time. We’re performing analysis; so therefore, we have detection limits of our analytical system. So if we have a poor analytical system — I’m not saying we do — but if we do have a poor one, the profile may be present but below the level of detectability. And then during the manipulations in the lab, we often, we will do differential extractions, and we do use multiple manipulations. So the more manipulations you do, the more chance you have of loss of analyte.

So for various reasons we have a problem. Some of the solutions to the problem we thought early on for [the] initial study was to use YSTRs. Obviously with YSTRs we can ignore any — in theory — we can ignore the female component of the swabs that we’re going to take. And in theory, as well, we could use no differential extraction. We could take a so-called “low copy number approach,” and that’s not the politically correct term anymore. But I still use it because it’s — I was at a meeting recently in Europe, and Peter Gill, from the Forensic Science Service, who’s the guy who coined the term low copy number approach, doesn’t use the term low copy number approach officially. But during discussions and talks all he uses is “LCN,” and he actually uses the same terminology. So a little template in approach, meaning that we may be able to analyze DNA that’s present, say, in less than 100 picograms. Could be less than that — 50 picograms or less of DNA. And the low copy number approach — there are different ways that we can improve the sensitivity — adding large quantities of DNA, for example, increase cycle number and a variety of other methods.

Then, what turned out to be critical is also different sampling. Now, there is a wide variety of sampling. Maybe Pat Speck can talk about this. There’s a wide variety of sampling processes that take place just now throughout the country. I know myself; I worked, as I said, for 30 years doing this, and I remember the first day of my job we used cervical sampling. It was a standard method. So many people do cervical sampling — cervicovaginal — some places don’t. It turns out in this particular study the ability to obtain a profile beyond four days, five days is probably critically dependent upon the cervical sampling. So what we did was we, when we started this work there was no commercial YSTR kits around, so we did a lot of this initial work with our own kits and our own samples and our own protocols. And so the conclusions of this “early work” I would call it, is that — again, not with standard kits and with our own conditions, and I’m not going to talk about this because I’m going to talk about more recent work — it’s possible to obtain a genetic profile of a semen donor in postcoital cervical vaginal samples, covered up to four days after intercourse. We used carefully selected YSTR markers; we performed new differential extraction; we used a large quantity of, therefore, of input DNA; we used increased cycle number; and we used cervicovaginal sampling. So that was initial work, and we published this initial paper in 2003.

So what happened is we kind of let it go for a while. Then we decided, OK, now there’s more interest because now there are commercial kits available. So we wanted to then — because obviously we didn’t expect many labs to be able to use their own kits and don’t want to anyway, with a notable exception of New York City, who were the first lab in the country to use YSTRs in casework, using their own primers, et cetera. So we did some work with commercial kits, and the studies I’m going to talk about now are postcoital samples taken from — these are controlled studies. And it’s important that we do these controlled studies because we want to be sure that if we say we’re detecting a male profile X number of days after the event, then it’s true. If you take real casework material, which we have to do at some stage, you’re basing that information about postcoital time on normally the victim. And some of these statements are not 100 percent correct. So there’s a certain element of inaccuracy in some of these — because of recollection, some of the victims may be under the influence of drink or drugs, that type of thing — especially these longer interval times.

So we took five donor couples, and what we did was we took cervicovaginal swabs, too, taken by each female volunteer three to seven days after separate acts of intercourse. So, three, four, five, six and seven days. And it would be only taken after a seven-day abstention period. So what I mean by that is that you would have couple one, they would abstain from sexual intercourse for a week, and then they would take a precoital sample. The next one down there, the next — the negative control is taken, that is a precoital swab is taken. That has to be negative. And then three days later, the victim will remove a cervical vaginal sample herself. Then in that same victim — not victim, sorry — the same donor ...

[Laughter.]

Ballantyne: My understanding is there wasn’t a victim in this case.

[Laughter.]

Ballantyne: The same person and the same couple will then do the same thing: abstain from sexual intercourse for a week, take a precoital sample, and then have sexual intercourse, and then four days later take another sample. So these are separate samplings. They’re not, for example, taking [a sample] after one act [at] three-day, and then will take another sample four-day, five-day, for obvious reasons. Because if you do that, you’re going to remove — you have to. Every contact leaves a trace, remember? I mean it’s like basic. You’re going to remove — and some people argue, “No you don’t.” That’s impossible, right? So we’re removing sample, if you go and take sampling at three days and four days and five days; so we have to do them separately. And we use the term “cervical brushing.” I think I’ve been hammered for this. It’s not really — and Pat’s probably hammered me, and her colleagues. This term is used; basically it’s just a cotton-tipped swab. It’s nothing special. And it’s basically sampling of the cervix using a brushing motion, apparently. So when we do this, here’s just some of the data because I’m trying to move on to other data. Here’s a four-day, I’m not showing the precoital — I’ll show you in some instances as time goes on probably, later dates.

Here’s a four-day sperm fraction — Yfiler — and here’s a full profile. And, of course, we’ve compared this to the known donors. This is a full profile. That was with Yfiler — he’s with PowerPlex Y, the two kits, right. The Promega kit and the — we looked at the Promega kit, the commercial kit and the Applied Biosystems kit. We also looked at the company in — what was it called again? In Louisiana. Reliagene. Because they’re not longer in existence. We also looked at Reliagene kits also. We reported that in our paper; I’ll show you. So four days we can get a result. And here’s a pre-five-day from one of the samples, and you can see there’s nothing there. And here’s a sperm fraction — now we, remember I talked that we didn’t do a differential; now we do differentials. We do a differential, and we actually prefer a differential now with the commercial kits. So here is a profile five days; this is five days after intercourse. And here’s the other kit.

Here’s a six-day sample. Now you can see in the six-day sample you have a partial profile here. But the profile, the alleles that are present — 12 out of 17 — are in fact the alleles from the male donor in this case. And this is standard conditions. These are standard. Now I should say the important thing about this is that the standard amplification conditions are used. This is not increased cycle number or anything else. Here’s another six-day sample. Here’s a six-day differential. So you can see that at six days you start to — five days we often get full profiles — six days we start to get partial profiles. And here’s a seven-day sample, and we don’t claim yet, believe it or not, although you can see on the right-hand side there are some allelic peaks there, and they’re consistent with the donor. But I would not hang my hat on that particular profile at this moment in time. But I’m just showing you this is what we’ve seen. And our aim is to keep going with this in terms of the number of days after intercourse.

What we did, we used a number of DNA profiling enhancement strategies. We called on that in a genetic sense. Cervical brushing is used. The differential, we prefer differential now, which is contrary to what we initially reported several years ago with our own kits. And I didn’t talk about this but we, in some cases, we will use post-PCR purification methods. Standard manufacture cycling condition was used.

The summary is that full profiles were typically easily obtained from these couples — it’s a limited study, remember, but nevertheless three to five days after intercourse. We can get profiles greater than or equal to six days, but they’re partial, mainly partial. And although I didn’t go into this because I don’t have time, the use of post-PCR purification significantly improved the ability to obtain a profile, especially from the five- to six-day sample. So that can be employed. To reiterate, again we found better profiles with differential lysis. And we still hang our hat on this seven-day profile, but we, again, we haven’t really — I can’t stand up here and say that seven days is a standard or routine method yet.

So we published this work in two papers. The top one is “Performance Characteristics of Commercial YSTR Multiplex Systems,” but that’s basically up to three days showing a simple and straightforward [method] to get routine, standard YSTR profiles three days after intercourse. And then the other paper, the bottom one in 2008 shows some of the data that I just described. So greater than or equal to three days we can get a YSTR profile.

So the current work we’re involved in now is that we’re actually specializing now in greater than or equal to five days. And we use a variety of strategies that are listed there. I don’t have time to go into them at the moment, but we are using a variety of strategies to try and improve our results. And then what we are also doing is revisiting the use of autosomal standard STR analysis just because we want to. It’s good fun.

[Laughter.]

Ballantyne: Because there are not that many studies on it. So we’ve been looking at certain things. For example, we have an alternative extraction method. Typically up until now we have not actually optimized the system where we take every single — although we collect our samples and extract DNA, we don’t actually add the whole lot, ever. We haven’t done that yet. So we know that we have room for maneuver, so to speak. So we’re looking at alternative methods at the moment where we can basically take DNA extracted and basically run the whole lot, every single piece of DNA. Remember, this is a controlled study, so we’re allowed to do that. All right, so in this particular case we’re looking at [a] particular silica-column-based purification method. The work is currently in progress, but we’re quite happy with the ability — the nice profiles — that’s two-day — here’s a two-day from this new method of extraction — three days. You can see there’s partials in some cases. Here’s a pre-six-day. Here’s a six-day. Again, it’s partial, but there are some alleles present. In fact, when we compare — and here’s a six-day sperm fraction. Another six-day sperm fraction that I previously showed you — and this is what we found. We found that if you take the six-day fraction with the standard so-called method and then we do another of this alternative extraction method, when we actually look at the results, basically — and you can’t see it very well in this I don’t think — but basically you see the results, first extraction, second extraction, and the bottom line is that on two separate occasions when you amplify this, you can get a composite profile. Now, again, I’m not suggesting that’s what we do in a real-life situation, but we can recover from that composite profile an almost complete profile.

The argument could be, it’s probably, it could be just stochastic; it’s a low copy number, so you amplify it once, you amplify it again. You get one allele coming up this time, and then [another] the next. Who knows at this moment in time, but we’re beginning to see that doing multiple amplifications may help in this type of thing.

We are looking at alternative enzymes. And one particular one that we’ve done recently, used recently is SuperTaq Plus from Applied Biosystems. And what we’re finding with this enzyme is that if you take the RFUs, we get an increase in the RFUs quite often. Some now increase, sometimes [there’s] no increase, but there’s an increase in [some of] the RFUs we found, certainly. So we’re interested in this. And here’s a Taq gold standard three-day and a SuperTaq Plus. I know it’s hard to see, but that’s a nicer profile, higher RFUs, et cetera.

To conclude I want to then talk about the autosomal, what we’re doing. So we’re just basically taking the same samples, same sampling strategy. And here’s autosomal, here’s from a four-day sperm fraction, using Profiler Plus in this case. You can see there, there’s a clear mixture. But the male profile is clear, is there. Now you have to de-convolute that mixture, but the male profile is clearly there. And it’s quite straightforward to de-convolute that mixture, relatively. Because we know the female, right? So that’s four-day. We’re finding [at] four days quite often that’s what you get. However, we hit the roadblock at five days. When five days, using the methods I’ve described, we just don’t seem to see any autosomal signal. But, remember, it’s a small number of samples, relatively, obviously. And it’s a small study. So we hit that roadblock. We know that with YSTRs we can go five, six days. With autosomal it seems four days in our hands, so far, that’s what we’re getting. And also, obviously, it becomes more difficult; you have to de-convolute that mixture in the autosomal setting.

And, basically to get this profile, we’ve had to use only one, do one wash of the sperm, et cetera, and these things I don’t have time to go into. The current and future work is that we want to continue with the postcoital samples greater and equal to five days and apply these variety of novel strategies that I didn’t really go into in any great detail, but there are a few of these tricks in our toolkit. Revisit use of autosomal STR analysis, because we really don’t want to stop at four days. We need to go beyond that. We need more extensive studies. We need more extensive studies, more samples, and hopefully — Pat Speck here will talk in a minute. We have set up a collaborative study with Pat Speck on paper. I don’t know if she’s going to talk about it, but the bottom line is we’re going to get large numbers of samples, if we can get funding for it. So we would build to do this with larger numbers of samples from real — are you going to talk about this, Pat? No? You are? So I’ll let her talk about it.

[Laughter.]

Ballantyne: I didn’t know. So, with that, I’d like to thank the — first of all I should have said that the person who has done all this work in the recent past is Erin Hanson, who is sitting in the front row there. All the other work was done by Ashley Hall, Pam Smith, [and] Kathy Press. And I’d like to thank NIJ for the funding and the support. And with that, thank you very much.

Patricia M. Speck, DNSc, Assistant Professor and Public Health Option Coordinator, College of Nursing, University of Tennessee Health Science Center, Memphis

Pat Speck: Will everybody sit down now? Hi, I’m Pat Speck from the University of Tennessee in Memphis. How many of you are going to have trouble with my accent?

[Laughter.]

Speck: Well, we’ll just get over that. But you can move closer. I also would like to thank Lois Tully and the National Institute of Justice for actually reaching out to the clinician community. Sexual assault nurse examiners have been in business since 1974. The program began in Memphis, Tenn., and it was the brainchild of a family nurse practitioner named Beverly Bounds. She wrote a federal grant and got it. And the program is up and running strong 35 years later.

The first program is not the only program. There are now well over 800 programs that employ nurses at a variety of levels. Now I mention that because the National Institute of Justice, as well as the Department of Justice, have spent considerable time and energy ensuring that victims have a say. And one of the ways to do that is to bring them to care that is therapeutic, coordinated and can bridge these systems that are so separate. One of the things we’re doing with Dr. Ballantyne is trying to bridge some of those systems. So I speak to you today as a clinician, as a person who is a family nurse practitioner. I have my own practice, and I’ve started in the SANE business — if you can call it that — in 1982, long before “forensic nurse” was the word. So if you understand that, you’ll know that I’ve seen quite a few victims. And I’d like to recognize Mike Hill, who’s here, who used to bring patients to me — in the back, back there — as an investigator in Memphis, Tenn., and he came up here. This has been a great reunion time.

So that said, let’s get going. The goal of this presentation is to inform the scientific community about what is known, and, more importantly, what is not known about factors that contribute to the successful collection of DNA, including issues associated with the rape event. What is that postcoital environment? What about evidence collection timing? We learned a little bit from Dr. Ballantyne that maybe we can extend some of these minutes to hours, and maybe even a day or two. What about the system-wide financial and legal ramifications of lengthening this timeframe? What’s it going to cost? I used to say in my early days, “please don’t identify every victim; we can’t handle them.” We were getting about 1 in 10 in the ’80s that would report to law enforcement. If we got 10 in 10, we would have been seeing over 3,000 patients a year. And we couldn’t handle it with our infrastructure. So all these things have ramifications. What about the timeframe? What about the criminal justice system? The more cases that go through, the more evidence that’s developed, the more cold hits that happen.

What about the SANE community? We have registered nurses and physicians who are working with these patients, but the laws govern their practice. And it’s in that legal ramification of the practice. This is a licensed profession — registered nursing, medical physician. The licensing requirements in states vary. So if you don’t know about that, you need to find out about it. What about the laboratory system? Can they handle the volume? And what about the victims that we all serve? What is their voice here?

So that’s my goal today, is to kind of cover that in about five minutes.

[Laugher.]

Speck: You know there are multiple conditions that contribute to the successful recovery of DNA. And that has changed over time. Some factors that influence that are known — for instance, the timeframe in evidence collection. However, in these new studies we’re looking at an expansion. But one of the discussions that occurred at our TWIG meeting was, “Well what about the timing at the month? What if it’s an older person? What if it’s a younger person? What if they’ve had a surgical hysterectomy? And what happens if, if, if, if ...” And before you knew it we had a lot of questions, more than we were answering with what’s happening in the laboratory.

So these concerns about increasing the costs, increasing law enforcement, increasing SANE time, possibly decrease the likelihood of recovery. And there are just too many questions we don’t know the answers to. I go back to my nurse practitioner practice, and I say, “OK, my infertile couple comes in. What am I going to do with that?”

So if you get a queasy stomach talking about some of the things you don’t talk about over the dinner table, you might want to consider walking out.

[Laughter.]

Speck: How many people here know what E.D. is?

[Laughter.]

Speck: You know it because of the advertisements, right? Well, you know what? In my area it means emergency department.

[Laughter.]

Speck: But today we’re talking about E.D., the erectile dysfunction and ejaculatory dysfunction. So automatically we’ve got an offender out there that may have these problems. OK, and may not give us anything but a few cells. Because, remember Locard’s theory — there’s cells exchanged. I once said to the group at TWIG, “Give me a cell in the vagina any day over a sperm, because sperm travels.” You can’t get the cell unless it’s shoved up in there. Think about it.

Condom use. We saw the advent of condoms being used in rape with the HIV epidemic. OK. And, of course, the famous case in Texas where a baggie was used and then she was accused of consenting because she asked him to wear a baggie. Go figure.

Pre-ejaculate. Most people don’t know that that’s kind of flowing out all of the time, and when it does it also ends up in the urine, which ends up in the underwear, which can — if it contacts — end up on the victim. And then there are other issues, too.

So in 1987 the first assailant was convicted of sexual assault with the help of DNA. And this case was upheld on appeal. They used RFLP. And those of you who know the history know that it used up every bit of the sample — every bit. So there was no challenge by anybody because it had to be used in RFLP. It took years for these laboratories to gear up, so we actually, in the center in Memphis, Tenn., started collecting for DNA in the middle ’80s because the science was out there, and we knew the evidence would be sitting in a police property room — dry, usually temperature-controlled. And then if the police could find it, they could bring it up. That actually came to fruit with the Innocence Project. By the way, that was not very successful in our area; only one person was freed out of over 40 they looked at.

OK. So, what did we know about it pre-1999? We knew that specimens could be positive for sperm. This is looking at the skin, the anus and the oral areas. And look, three hours we could get a very positive skin sample. And then after five hours it dropped. I promise you since that’s a skin sample at eight, it was in a crack or crevice that wasn’t going to be washed away. In the vagina it was even worse because you get samples, but they go down very quickly. Two hours, three hours, and then you get out here at 39 hours and very, very few people had a positive semen sample. Now what was going on there? Because we know from Jack’s work that, what, six days out we should be getting something, but here we are, just 39 hours out. So what’s the difference? Could it be the difference in what the victim’s development is? What their health status is? What their reproductive status is? And so these are questions that we have to ask.

And in 1999, Dr. [Linda] Ledray said these data clearly indicate the vaginal site is most likely to obtain specimens positive for sperm. Well, those of us in the infertility area knew that the cervix was the best place and usually at menses — I mean at ovulation, so that you get a real thick cervical mucus, also called the “spinbarkeit.” And this cervical mucus just loves sperm, so if you can find that then you hit the jackpot because it’s always going to have sperm in it. But they have to be at that time of the month, and they also have to be ovulating. What happens if you have any of those women who are not ovulating? Maybe on their menses? Maybe too old? Or too young? So all of these issues came to my mind, and I said, “Maybe this is another area we need to ask more about.”

Of course there’s some winds of change, and that is that forensic science is under fire. And Discover magazine said it’s quite bad. A new understanding about the reproductive track has to occur in the forensic community. In the historically health community, we get the health track. We know what health is all about. But the forensic community has not looked at health as a variable. What about the aging? What about the development? The forensic community hasn’t considered that at all because in the laboratory, you get the sperm, and that’s where you are. And it’s OK; it’s just that’s where the discipline is.

We have to re-evaluate the literature related to 72-hour evidence. And we’re going to look for strong community partnerships, and that’s exactly what I think the vision of NIJ was. Putting a clinician in with the laboratory directors was really a gold mine of exchange. And so partnerships are important.

The infertility literature — there’s lots of information about sperm. Let’s talk about it. Normal men have normal sperm. Normal men also have sperm that are not-so-normal, and it could be for a variety of reasons. But let’s talk about the normal sperm: 42 to 76 days from the time the sperm produces, it comes out in an ejaculate. Now, if it’s in the process that means about half of those guys that got out there too early are already dead. And the ones that are too young are not real fertile. So you really just have this kind of bell curve of sperm that are viable in the center. Fertile life span is 24 to 48 hours. Male human ejaculate is only about a teaspoon, although many men will tell you it’s much more.

[Laughter.]

Speck: There’s about a million sperm there that are viable. And about 20 million sperm, unless you’re going to be infertile, for the most part.

Low sperm count. A fellow wearing brief shorts all the time, working in Key West is going to probably have a lower sperm count. That’s because the cremaster muscle can’t work, which means it’s too close to the body; it’s too hot, and the sperm die off. So briefs, out; shorts, in.

Motility. Motility is dependent on the environment it’s deposited. Normal sperm morphology is critical. Sperm can have an oval head, a round head, a little mushroom cap. It looks different in different places. And there are structural problems that can contribute to whether or not sperm can be detected. So if the testes don’t descend or if the sperm are deposited in an area in the vagina that’s not the place where it’s expected to go, which is the very distant, posterior fornix, and maybe it comes out in the mid-vagina because the urethral opening is on the penis and not at the tip, then there’s a problem.

Blockage in the tubes that transport sperm. Have a guy that’s had a bunch of STIs — he’s going to be infertile, just like the woman who’s had an STI or two in the tubes is going to be infertile. Varicoceles are like veins on the legs. So if you have a variocele, that’s also going to create some problems with the sperm.

The reality is unless you have done a medical exam on the offender, you can’t know the quality and character of his sperm. How many they are? What time of the month it is? Whether he wears briefs or shorts? All of these things may impact whether or not he gets a good 100 million sperm out there. That’s the ideal. Get me that 100 million, and I’m going to give you a good PCR, STR, YSTR outcome.

Now, let’s look at the vaginal environment. Where is that patient in their cycle? Are they still having cycles? What about if they’re before menses? What about peri-menopausal? What about menopausal women? What is the menstrual cycle timing? The chance of a woman becoming pregnant at any time in a month is 1 in 30. There’s 30 days. You wash out the lining. There’s only one day essentially that that sperm can penetrate. There are some women that are very unlucky.
[Laughter.]

Speck: Existing conditions. What happens if they have fallopian tube disease? What happens if they have endometriosis? What happens if they have any number of other things that are going on that you can’t control for?

And then what about the medications that they’re on? I didn’t mention that about men, but if they’re doing medications it affects them also. Just don’t drink and take Viagra. Don’t take nitroglycerin; it lowers your blood pressure. So the effect of the medications on the genital system are — and I’ll give you an example: Robitussin. How many of you love Robitussin when you have a cold? It’s great. It causes the cervical mucus to become liquid and contributes to infertility. So there are medications that actually cause problems.

The other thing you have to know about is that it’s not only Venus and Mars, but there’s a battle when sperm gets into the vagina. The vagina considers sperm a hostile invader, and it’s in this hostility that the sperm actually says, “I can overcome this hostility.” So, what it does is the bolus creates a gel that forms within minutes. And if it’s deposited in the right place, which is by the cervix, that’s going to last for about two to four hours. And then there’s a thing called “flow back.” And that happens because those guys, they just can’t hold out, so they get liquefied. When that happens it drains out of the vagina. That’s called flow back. Thirty-five percent of sperm is lost in flow back within five to 120 minutes.

So guess what happens to your capacity to recover sperm? Where is that sperm? It’s on the panties. So be sure and look at the panties because that’s where it is. Very few of those sperm enter the cervical canal because they got to swim in there. So the ones that are closest to that cervix have the best opportunity to get into that cervix.

How many of you know that the hymen ages? How many of you, first, know what a hymen is?

[Laughter.]

Speck: For those of you who don’t know, that’s the “cherry” that men have this fantasy about.

[Laughter.]

Speck: The hymen is a connective tissue that covers like a collar. It goes around the opening to the vagina. Sometimes it covers it completely — that’s abnormal. Sometimes it has multiple holes in it. But it ages. It is very thick at birth. It thins out and becomes very, very thin where you can see blood vessels in it. Then it starts thickening again. And it thickens its most during pregnancy. Then it starts thinning again until the little old ladies complain of the men who are taking Viagra that they’re married to. Because they don’t want to do it and the guys do. And they don’t want to do it because they’re too dry; it’s too thin; it hurts. They haven’t realized if they just go in to their healthcare provider, you can get some medicine to fix that.

So the postcoital sample that’s found up to seven days using the YSTR is found in reproductive women who have gone to the right spots. They have I assume — and this is an assumption — normal men they’re having intercourse with, and so you’ve got the ideal sample here. Cervical swabs will produce sperm when vaginal swabs do not. A nurse took some of the stuff that we’ve been talking about, and she looked retroactively into her data, and she looked at the cervical swabs they collected and the vaginal swabs. And her name’s Jean Morgan out of Ohio, and she found that guess what? We find that in the cervix more often than we do in the vagina. But don’t forget those panties.

We have a “model for information explaining and future understanding” of recovery of DNA. And this is called MIEFU just because I like acronyms. Victim plus assailant means time and activities, and that will all impact your DNA recovery. It is not independent. It’s all over the place; we just don’t know where it’s all over the place. So victim factors will influence recovery like cellular material, whether it’s sperm or cells. The assailant factors will influence the quality and quantity of the cellular material deposited. And the time will impact the degradation process. So the further away you are from it, the more likely you are not to be able to recover it. And the reality is that we don’t know many of those factors.

So let’s talk about research rate timing. The incidence of delayed reporters. We know that there’s a possibility that those women who cannot come forward in a victim-centered system may actually have an opportunity to come forward three or four days later. And that’s important because it supports the healing.

We also know the prevalence is going to be impacted. Epidemiology is going to be impacted. We’ve got reimbursement strategies, personnel costs, supply costs. Collaboration. How are you going to get an entire team to buy into collecting? What kind of investigative costs is it going to be if an officer has to go out on a rape that’s five days old?

What’s your community experience? What’s your lab doing? Because if your lab is actually collecting after 72 hours, are they, in fact, finding this sperm? If they are not, don’t change your protocol yet, because it may be that we’re going to end up with a tool that says, “If you have A, B and C, you collect; if you have D, E and F, you’re out of luck.” So unless new procedures and equipment, qualified personnel arrive, or your community experiences, we hope to provide the cohort in this proposal that we’ve submitted as a collaborative to really get the group of people.

Let’s talk about it. It’s a collaborative of the University of Tennessee and the University of Central Florida. We have applied to the NIJ, and we’re going to use the YSTR, even though it’s not maybe used in all the laboratories, it’s the common thing. And it’s because it takes mixed samples, and way too many rapes are multiple assailants, so we’ve got to be able to distinguish. You need about 100 forensic nurses. They all know where to go to get the evidence. They know where their cervixes are, and we expect them to volunteer because they all wanted to give up their bodies for forensic science. We also want to pay them. And if you know of a forensic nurse, you can certainly be part of this process, and we’d be really happy to have you as a volunteer, too. There’ll be certain criteria that need to be met. Obviously, building on what Dr. Ballantyne has started in terms of collection and timing from the event.

So I hope you enjoy the picture. Thank you very much.

Mechthild Prinz, Director, Department of Forensic Biology, Office of the Chief Medical Examiner, New York, N.Y.

Mechthild Prinz: I want to thank Lois for inviting me to speak on this panel. And I know there’s many, many people in this room that could give the same presentation. I’m supposed to present a practitioner’s perspective, and I know for a fact — you can’t hear? Now? OK. All right. I thought you were volunteering to give your perspective.

[Laughter.]

Prinz: I don’t want to be stating the obvious to a group of colleagues that are many of whom I’ve known for many, many years, and I know you all know what the impact would be if we received an increased volume of kits. And I almost came without a PowerPoint because I thought it was more of a sit-down panel, question-answer session. Actually I have to ask you some questions, and I have a lot of question marks on my slides. So, increase in submissions, what would that mean, and how would a more sensitive, new test modify the work flow? Increase in DNA tests, what would it mean? The reporting and even the testimony might be affected.

So, first question is if we go into these really extended postcoital intervals, will our current screening methods still work? The screening methods we use for semen-like alkaline phosphatase, p30 ELISA, sperm surge, those strip assays, I always forget the names. So, would this still work for a seven-day-old vaginal swab? And if no, would you just skip all the semen screening and go straight for male quantitation? Can I have a show of hands, how many laboratories are using a male-specific quantitation right now? So that’s definitely quite a few. How many of you go straight to extraction and do not even do semen screening anymore? All right. We’ve got one. So then the question is do you do that for all sample types? Do [you do it] like for a bed sheet — where we do the A.P. mapping to find the semen stains — or only for the kit items?

Audience member: Swabs.

Prinz: Just the swabs. So, let’s say you would switch your protocol to skip the screening, you cannot take that method offline because you still want to use it for other items, like clothing or bedding, but you might change your work flow for the rape kits only. Like some labs don’t do A.P. on kits. It’s something that you need to think about, and definitely to have the male quantitation online does help you to make an informed decision on where to go next with the sample.

I think, aside from the YSTR test, I think being at this meeting made it very clear to me that the extended intervals will become feasible because there are new methods out there. There’s this laser micro-dissection that can be used on samples where sperm is old and degraded, and there are very few. There are new, potentially really, really useful laser capture chemistries out there that might help to get the few sperm cells more consistently recovered. Even the autosomal kits — and I think Jack already showed the data for that — have gotten more sensitive. The companies are putting secret ingredients in the buffers and adding a cycle here and there. And for example, a MiniFiler kit now has 30 cycles. And they were also in the research section, there were repair enzyme posters or better tacks. So if the tests get more sensitive and we get better at recovering sperm, that definitely means we would anticipate a higher success rate on a collected vaginal swab. But I’m with Pat — do not forget the panties.

[Laughter.]

Prinz: I do not know, and I’m not sure anybody knows how many cases do get reported after the 36-hour interval that we feel is a good cutoff. So I have no idea how big the backlog would be. I have some numbers for New York City. This is just for one quarter, the third quarter of 2008. The rape percentage has kind of stayed constant over a couple of years now; it’s about 1,500 per year. So in that quarter, we did receive 375. And in our hands, 47 percent of the kits are negative for semen. And we use p30 ELISA to determine that. And if a kit is negative for semen, based on a serology test or ELISA test, one of the entry-level criminalists can write up the report. If I don’t do the semen test but just do a straightforward male quant to determine male DNA is present, yes or no, it has to be a DNA-qualified staff member that has to write the report. Because as soon as you have real-time DNA data, you need molecular biology, biochemistry, genetics continuing education — you have to be an interpreting DNA analyst. So it has a potential impact on the staff structure.

The 47-percent negative kits — what our next step then of course would be [to] look at additional items, such as panties, clothing, anything that has been submitted that might have potential semen on it, tissue samples, and things like that. So we already know that the fact that the kit is negative doesn’t mean this doesn’t wind up to be a DNA case. And 200, that’s why the 176 and the 213 don’t add up to 375. So that’s the explanation; that’s the additional evidence.

Another show of hands. How many labs do Y’s? Yes, yes. I’m very happy to see that. The YSTRs already increase the number of samples that are feasible for DNA testing because it is really, really useful in all cases where you have saliva as a biological fluid on an intimate sample like a vaginal swab. And it also pulls up the male component if you did the autosomal test, and you have minor, minor, minor male peaks, you get a nice, strong Y signal if you go to Y’s. So approximately 10 percent of the rape cases in our lab actually do go to some type of Y testing. So we already did experience an increase in samples just through this technology. The Y’s are obviously limited because there’s a reduced statistical significance of the haplotype, and you can’t use it in CODIS. Our main goal on a stranger case, of course, is CODIS. And then if you do have a male-to-male mixture, and it’s 1-to-1 in the ratio, then you have a real problem with the statistics.

The male-to-male mixture is something that we actually might see more of if we start collecting in extended intervals. Because the chances that the victim had consensual sex, of course, increases the longer — the later we collect after the — that she had seven days prior maybe had something, so I think that would increase. Hand?

Pat Speck: She could have sex before ...

Prinz: Right.

Speck: ... with her partner, have the rape, and then have sex after with her partner ...

Prinz: Right, right. And ...

Speck: ... you’ve got mixed samples.

Prinz: Yes, I think we would see more mixed samples. New York state did switch to cervical collection in the sexual assault kits now. We don’t have any data on it yet, but that also might result in having more mixed samples now. Jack mentions that already. Maybe, let’s say, Roger already doesn’t do semen testing on the vaginal swabs, and I know that some private labs don’t do that either. Let’s say, you would decide, “Oh, I will skip the semen testing if I know the kit is collected five days after the incident, but I’ll do it on the younger ones.” For example, our saliva test, we have a life span like that, where we know if a sample is too old we won’t even try. But then you need reliable case information, and as Jack pointed out, the victim might not be able to actually supply the exact time of when the incident happened. Then, do you want to make a decision? Are you going for differentializes or non-differentializes based on the interval information that you have? That’s a problem. Or would you do a different work flow if you have a stranger case versus an acquaintance case because if it’s a stranger case and it’s seven days, and then you might say, “Oh, Y’s will be the only thing that works, and that doesn’t work for CODIS. Let’s de-prioritize that case and not work it.”

I told you I had a lot of question marks in my slideshow. And these are things that we would have to consider, but I’m looking forward to the results of the study Pat talked about. I do think it’s probably coming because the technology has gotten much more sensitive.

I have been approached about semen results in court. So if you, let’s say, now you have this seven-day-old kit, you find a nice profile but you could not confirm that this male profile actually is from sperm, because you couldn’t confirm the presence of sperm. Will that still be a rape? Or will there be some type of very original explanation how the male DNA was deposited? I don’t think it’s a problem in New York City. I discussed it with some ADAs. It’s very rare whether that would make a difference, but I remember a discussion that we had in a different panel and other jurisdictions definitely had an issue with that. They really felt that it was very important that the criminalist can testify that semen was found. And then it would have that sexual connotation.

So I’m not sure. Does anybody here have experience with that? Any lawyers in the room? If there are, they’re hiding.

[Laughter.]

Prinz: I’m already finished with my presentation because I’m looking forward to having some questions and answers here. Thank you.