Fourth Annual DNA Grantees' Workshop

Monday, June 23, 2003

AFTERNOON SESSION

Practitioner Session #1: Systematic and Rational Approaches to Improve Forensic Practice, Method Harmonization, and Education and Training Standards in Forensic Science
Carl M. Selavka, Ph.D., Moderator
Biography

DR. SELAVKA: Thank you very much. My name is Carl Selavka. I'm the senior program manager in the National Institute of Justice's Investigative Forensic Sciences Division. I oversee the Crime Lab Improvement Program earmarks, which is a $44 million program this year with 43 lucky recipients, three of whom are from the Forensic Resource Network. I've been there for 7 months and 19 days as an accidental tourist from the Massachusetts State Police Crime Laboratory System, of which we have a couple of representatives here today.

As I was looking over the program today, I was trying to figure out what ties together the four presenters in this next session. The one thing that really sets them apart from the rest of the program is that they're all going to be talking about ways of providing systematic and rational approaches to improve forensic practice, method harmonization, and education and training standards in forensic science. All of them are going to bear something about how we can do better in every laboratory, in every research and development environment, and in every application of training and education down the road.

Streamlined Processing of Sexual Assault Kits: Progress and Early Applications
Jack Ballantyne
Biography

DR. SELAVKA: So without further ado, I'd like to give you our first presenter. Dr. Jack Ballantyne is an associate professor in the Department of Chemistry at the University of Central Florida (UCF). He's also the associate director of the National Center for Forensic Science (NCFS), a Forensic Resource Network partner at UCF.

Most important, I think we can honestly sum up his career so far as: This is a person who has demonstrated excellence as a practitioner, researcher, educator, and leader in forensic science. I give you Dr. Jack Ballantyne. Ballantyne: Slide 1

DR. BALLANTYNE: I'm here today as a representative of the National Center for Forensic Science, and I think that a word for my sponsor is in order. NCFS's vision is to provide proactive and innovative solutions to meet the challenges facing the investigative, forensic science, and criminal justice communities. It does this by providing research, education, training, and tools and technologies in three areas: biological evidence, physical evidence, and digital evidence. Since I know nothing about digital evidence or physical evidence, I will then be talking about biological evidence. Ballantyne: Slide 2

Our mission at NCFS is to assist the national and international forensic community by doing various things: conducting research, validating methods, providing operational support, and providing rigorous educational programs. Ballantyne: Slide 3

There are many things to think about when it comes to forensic biology. We obviously want quicker, cheaper, and smaller approaches to forensic biology. But typically, that's not the approach we've taken at NCFS. We normally have been trying to improve the ability to make methods better or to look for methods that will enhance our current capability. We haven't really concentrated on trying to make the current methods quicker, cheaper, and smaller. Ballantyne: Slide 4

Now, in terms of what I'm going to talk about today, a potential method for a speedy rape kit would go into the realm of trying to make things quicker and more efficient. We have several projects, and the projects are listed here: Y-chromosome markers, assessment and repair of damaged DNA templates, and so on. Ballantyne: Slide 5

I'm going to be referring to Y-chromosome markers and Y profiling because I'm going to leverage this to the problem at hand. Here's the problem: The current backlog of unexamined rape kits is estimated to be a very large number, and it's a significant public health issue. Women are being raped, and much of that evidence is either not examined or not examined in a timely manner; therefore, many rapists, who may otherwise be identified, are able to perpetrate additional crimes. Ballantyne: Slide 6

In order to reduce or eliminate that backlog, a number of different approaches could be taken, one of which is development of quicker and cheaper assays. Perhaps we can do something in the lab, but there are some other things that have to be done too, including applying more innovative case-management techniques and increasing the hiring and training of suitably qualified personnel. I'm only going to talk about the assay side.

Screening of rape kit evidence typically requires evaluation of intimate swabs for the presence of seminal fluid. Labs that test for the presence of sperm use labor-intensive, time-consuming microscopic methods. Particularly, swabs that are negative for the presence of sperm require a disproportionate amount of the examiner's time to process. Ballantyne: Slide 7

So how many people in this room still manage, supervise, or screen rape kit evidence for the presence of spermatozoa on microscope slides? How many people do this? (A show of hands.) How many people would like to have a fast method to do that? (A show of hands.) This whole concept hasn't been funded yet, but we're about to do the work.

Alternative approaches to using microscopic methods include use of ELISA (enzyme-linked immunosorbent assay), ABA cards, and so on. But such nonsperm methods do not provide reliable estimates of the amount of DNA available for analysis, and false positives, which are the biggest problem, can occur.

So my office has decided to develop a rape kit screening method that permits the rapid screening of vaginal, oral, and anal swabs for the presence or absence of male DNA. The concept is to simultaneously positively identify all body fluids present on the swabs and, if present, provide a male haplotype profile of the male donor at the same time. This process should take less than an average workday—6 hours, in theory—and will provide a screening method that allows a decision to be made as to whether to further process the samples for routine autosomal STR (short tandem repeat) profiling, because it's no secret that we'll be using Y–STRs in this approach. Ballantyne: Slide 8

So the aims were a rapid screening of swabs for the presence or absence of male DNA, positively identifying all body fluids, and providing a male haplotype profile of the male donor or, alternatively, indicating the presence of male DNA. They are the two approaches. The process should take less than 6 hours and identify a subset of swabs for routine processing. Ballantyne: Slide 9

We have been developing a number of Y–STR markers for a period of time now, and not all Y–STRs are equal. Because of the close homology between the X and Y chromosome, many Y–STRs will cross-react with female DNA under extreme conditions, where you have very, very large quantities of female DNA.

Here's an example of a multiplex that we developed in house with no differential extraction (i.e., we took 450 nanograms of DNA from a nondifferentially extracted swab and obtained a profile) after 48 hours. These have been judiciously chosen and designed in such a way that this would happen. Ballantyne: Slide 10

Here's another multiplex doing the same thing after 48 hours. Ballantyne: Slide 11

However, these particular multiplexes fail when you have a postcoital interval extended beyond 72 hours. Ballantyne: Slide 12

So we modified these to what we now call multiplex A (MPA) and B (MPB), a subset of these loci that allows the detection of 30 picograms of single-source male DNA in 3,000-fold excess of female DNA. Ballantyne: Slide 13

Using a 4-day postcoital sample, no differential extraction, and 400 nanograms of DNA, you get a male profile—MPA. Ballantyne: Slide 14

A similar thing happens with MPB, where with 30 picograms, no differential extraction, and 3,000-fold excess female DNA, after 4 days, you can get a profile. Ballantyne: Slides 15 and 16

By judiciously choosing these Y–STRs that work under these extreme conditions when you can have large quantities of DNA, one could perform the following: Take a swab and perform a direct lysis method; that is, you take a portion of the swab or the whole swab—depending, we have to discuss that—containing several hundred nanograms of DNA, do direct lysis, and then do Y–STRs, PCR prep, amplification, capillary electrophoresis, and so on. This should take approximately 5 hours and 40 minutes. Ballantyne: Slide 17

The theoretical approach is direct lysis to release DNA and RNA (ribosomal nucleic acid). (We'll come to RNA if I've got time, but I probably won't.) Then we coextract the DNA and the RNA together, we perform Y–STR analysis using these systems, which works well, or, as an alternative, using real-time PCR assay for the presence of Y chromosome, which is a bit like Eric Buel's discussion this morning where you could just look for the presence of Y. Ballantyne: Slide 18

But we would rather approach it this way, and getting a profile at the same time can be useful for identification purposes later. Then, we have a body fluid identification side of things, where we look at the RNA profile obtained from the RNA extracted from direct lysis to obtain the nature of the body fluids present on the swabs. So, for example, if it's a vaginal swab, it will confirm whether or not it's a vaginal swab, and it will state there's blood and saliva present.

Here is some preliminary work: A 12-hour postcoital cervicovaginal swab direct lysis. The direct lysis is the area that we're working on to try and improve the sensitivity and specificity of that, because that's the critical stage in this analysis in terms of making things faster. Ballantyne: Slides 19 and 20

An adjunct to this, of course, is to obtain the body fluid identification, and for this we're talking about RNA. Here we have DNA producing RNA to protein, and you also have the genotype, which is just the genome, but of course only parts of this genome are expressed in any particular cell, and the patterns of gene expression are unique to each cell type. Ballantyne: Slide 21

For example, blood should have a unique messenger RNA profile, and saliva should have a unique messenger RNA profile, what we have called a multicellular transcriptome. The complement of all RNA present in a single cell is called a transcriptome, but we are looking at tissues, different cell types, and multiple cell types in any of the body fluids we're interested in. For example, blood has contributions from several different tissues, so it's a multicellular transcriptome. Basically, we're looking for RNA, which is tissue specific. There are housekeeping genes, of course, but we're looking for tissue-specific RNA molecules that are abundantly expressed, because RNA is expressed at different levels. Ballantyne: Slide 22

To reiterate, the concept is swab cutting, DNA and RNA coextracted, and no differential extraction. There's a DNA side that looks at Y–STRs using a set of carefully chosen Y–STRs. Male profile? Yes. Process the swab with no further action. Ballantyne: Slide 23

At the same time, we have an RNA multiplex real-time PCR that gives us the body fluid identification, and so you have a male profile and the body fluids are present. That's the concept, and it can be done in less than 6 hours.

I want to just skip through this [slides 24–27] because we have identified a number of RNA molecules that are specific to the tissues we're interested in. [Note: Despite not being discussed at the conference, slides 24–27 are included in this online presentation.] Ballantyne: Slides 24–27

Here's a list of genes expressed exclusively in saliva, at least to our knowledge, and compared to all the other body fluids we're interested in so we can detect these RNAs. Ballantyne: Slide 28

These are the RNAs here, detected in five different individuals, and here are pictures of the RNA molecule. Ballantyne: Slides 29 and 30

So this is the proposed schedule:

• Develop and optimize the direct LYSIS method, selecting the quickest and most sensitive procedure.

• Develop real-time PCR assays for body fluid identification and male-specific sequences. We have the genes, so it's just a matter of converting to the real-time PCR format.

• Complete the optimization of Y–STR multiplexes through in-house systems or commercial kits. We haven't evaluated the commercial kits yet under these extreme situations because, hopefully, if they work, they would be fine, too.

• Complete the development of real-time PCR assays for body fluid identification and male-specific sequences.

• Optimize the system with mock case samples.

• Test the system with bona fide case samples.

• Submit manuscript.

• Develop procedures. Ballantyne: Slide 31

Thank you. Ballantyne: Slide 32

DR. SELAVKA: If there's a question or two, we can take it now.

QUESTION: How stable is the RNA?

DR. BALLANTYNE: It's extremely stable. What do I mean by extremely stable? People have a misconception on the stability of RNA. RNA itself is stable. Otherwise life wouldn't exist as we know it today. The problem is the nucleuses that are present.

Therefore, we hypothesize the following: Say that blood, semen, or saliva leaves the body and starts to dry. During that time, there's some RNA degradation taking place, but the dehydrated state should afford some protection. When you go to rehydrate the RNA, you simply go in with extremely strong RNAse (ribonuclease) inhibitors, for which the very, very good ones (the strongest you can get) are commercially available.

When we do that, we can detect RNA in stains for 9 months or longer. So the RNA is stable. Depending on which ones you choose, they also are very sensitive. I had some of the data, but I just didn't have time to show it. Overall, the chemical instability of RNA is a concept that's overemphasized. It's a pretty stable molecule but obviously less stable than DNA.

QUESTION: Your method is good, but as to the quantitation . . . (inaudible).

DR. BALLANTYNE: Yes, that's an issue. We say this is a screening method, or intermediately, a real-time PCR quantitation method, as was described earlier today. But we envision this as being a screening method, so there's no need to quantitate for a screening method, right? Now, if you were to use the data (the male haplotype profile), that raises some issues. So we absolutely have to think about that.

Thanks.

DR. SELAVKA: Thank you very much.