Thank you everyone for joining us today for our webinar titled Crime Lab Analysis of a Sexual Assault Evidence Kit from Arrival at Crime Lab to CODIS Entry. At this time, I'd like to offer a brief disclaimer that this presentation was produced by the Idaho State Police Forensic Nurses in collaboration with the IAFN through an OVC grant. The opinions, findings and conclusions and recommendations expressed in this presentation are of those of the contributor and do not necessarily represent the official position of the policy or policies of the US Department of Justice. This presentation is being presented by Ryleen Nolan, the Idaho State Police Forensic Nurse, along with her colleagues, Deb Wetherill and Matthew Gamet. At this time, I'd like to hand over the mic to Ryleen and she can introduce herself and present her presentation. Good afternoon, everybody. Thank you for joining us. Thank you, Shea, for the introduction. As she said, I'm Ryleen Nolan. I'm the lab manager for the Idaho State Police Forensic Services Laboratory in Meridian, Idaho. Joining me today is our laboratory system director, Matthew Gamet, as well as our SANE SART nurse coordinator for the state of Idaho, Deb Wetherill. So we're going to present a video right now that basically takes you inside our laboratory and walks you through processing of the kit. At the end, we'll come back live and talk about some advancing technologies, as well as have time for questions. So let me see if I can get this to work. Hello, welcome to the Idaho State Police Forensic Services Laboratory. My name is Ryleen Nolan. I am the lab manager here in Meridian. And today we're going to talk about crime scene processing of sexual assault kits from the time they are submitted through completion of analysis. But first, I want to start off with talking about what happens prior to that. In Idaho, we have sexual assault kit legislation. This legislation mandates what kits are submitted to the laboratory, when they're submitted to the laboratory, as well as timeframes for testing in the laboratory. Many other states also have sexual assault kit legislation and it's important to know what legislation is in your state and how that impacts you as a health care provider. The other thing I want to talk about prior to the kit coming to the laboratory is the laboratory itself. Here in Idaho, we are a laboratory system. We have three laboratories. However, all sexual assault kits come to this one location. That may not be the same for your area of the country or the jurisdiction you work in. There are county crime labs, city crime labs, as well as state labs. And depending on where you're located will depend on where your kit is sent. Laboratories, most of them that process sexual assault kits and perform DNA analysis are accredited laboratories. What that means is they must adhere to a specific set of standards. In our case, it's the ISO 17025 International Standards. There's an accrediting body that will look at the laboratory, look at all of their methods, look at their facilities, their security measures, how they handle evidence, and ensure that they're meeting the requirements of those standards. This is important to make sure that the laboratories analysis and testing can be presented in court and have the strength of accreditation behind it. Another thing in Idaho is we require certification for all of our analysts. They must be certified in their primary discipline. For our analysts that are examining sexual assault kits and performing testing on those items, that accreditation is through the American Board of Criminalistics and it is the DNA or molecular biology accreditation. All of these things are things we do in Idaho and they may do in your state as well, just to ensure our scientists are at the highest level possible and performing appropriate analysis on these sexual assault kits that come into the laboratory. Most laboratories have what's known as a LIMS system or Laboratory Information Management system. It's simply a computer program that allows us to track our evidence, track the analysis that's performed, maintain chain of custody. Here in Idaho, we have a pre-log system, meaning our agencies actually log their evidence in from their office before they send it in. We accept evidence in person, which would be the same if you are a city crime lab or a sheriff's office crime lab. That will be the same. Evidence will be submitted in person. We also accept evidence via carrier or courier. It could be UPS, it could be Federal Express. This would be the same if your state has one centralized crime lab like we do. When the evidence comes in, we have a forensic evidence specialist and this person is responsible for ensuring that the documentation that's received with the evidence is correct and matches the items that are submitted. They also are looking to make sure the items are in a sealed condition. This is important for maintaining proper chain of custody. Chain of custody is the record of an item, where it's been, who's had it, on what dates, throughout the lifetime of that item. It's important that that be maintained so the integrity of the item is assured for court purposes. We also have a sexual assault kit tracking system and I know many other states have this as well. This was developed for our state in 2016 as a way of determining what kits were in the state of Idaho, what kits had been tested, what the status of those were. The tracking system in general are tracking them from the time a kit is sent to the medical provider through destruction of that kit. Destruction is typically mandated by legislation or an agency's own policies. We also are using it to track statistics and it can be used, the one we use, a victim will be given their kit number and they can go in and track the status of their kit where it is at in the process. So today we have one of our forensic evidence specialists who's going to be receiving a kit in. So what she's going to do is be comparing the information on the paperwork with what's on the kit. She's then going to log it into our system and assign it a barcode. So our LEM system will give information that the agency has entered into the system, including their agency, their agency case number, the item number, and specific to sexual assault kits, that sexual assault kit number that we talked about. It is actually barcoded on the top of the box itself. So this is the sexual assault kit for the state of Idaho. All kits are going to be different. Some have a larger box, some may have a different shaped box, some may also be an envelope. But usually the contents are fairly similar from state to state. Idaho does not have room in our kit for clothing. We ask that that be submitted separately. Typically, if your state has a larger box, they will be asking you to include some items of clothing. Once that kit is logged in, a barcode is placed on the kit, and this can now be used for scanning that from location to location, which is how we maintain our internal chain of custody. The next step would be to store the kit until it's ready for a scientist to check it out for processing. Sexual assault kits are typically stored refrigerated or frozen. If there's liquid blood in the kit, you would not want to store it refrigerated because it's possible when freezing that blood tube could break open. If there's not liquid blood, it can be stored frozen. They can also be stored at room temperature as long as it is in a temperature-controlled environment, and that will maintain the DNA analysis. Now that our evidence has been received into the laboratory and we've discussed all of the issues like legislation that can impact that, we are now moving into our DNA laboratory. Here in Idaho, our laboratory is made up of approximately 15 scientists. Compared to some of your states, that may be very small, and compared to some states, that may be very large. When I started here at ISP 20 years ago, the laboratory was two people, including myself, so we've grown significantly over the years. So, again, it can be any number of individuals that will comprise your state from two to hundreds of DNA analysts. When a DNA analyst comes into the laboratory, they've obtained the evidence. This is our kit that we accepted earlier and brought it back to the laboratory. The first step is to prepare themselves. A lab coat, hair pulled back, gloves put on. I normally have a mask on at all times when doing analysis. However, for today, for ease of you understanding what I'm saying, I will not be wearing my mask as this is not an actual item of evidence. The next step is to prepare a work area. The surface needs to be sterilized. We use bleach. Here in our laboratory, we put down clean paper and get all of the tools that we will be needing to do the analysis. We have workstations set up with all of this prepared for depending on the type of testing that will be done. We're set up here today for ease of filming. As I mentioned up front, we have our laboratory information management system. We're paperless. That may vary from state to state. They may still take some paper notes. They may be all electronic. So I'm going to be taking my notes electronically. It's important that the scientists record their notes as they are looking at the item. That ensures that the note-taking is accurate, that they're looking at the actual item of evidence when recording those notes. The first thing I'm going to do is ensure that it is sealed and the seals are intact. I'm going to look at that chain of custody and make sure it was signed into our laboratory. I'll then be looking at all of the information on the box and making sure that that matches the information in our system. Once I've done that, I'm going to mark the item of evidence with my date and initials. This shows that I had the item in my custody in addition to that electronic chain. But the reason we mark the outside of the box is when this is handed to me in court and I'm asked if I recognize it, I can immediately look for my date and initials on the box. Once I've done that, I've assured that the item is in a sealed condition. The chain of custody shows it coming into our laboratory. The information matches. I'm then going to open the box. The sexual assault kits here in Idaho, again, they will differ from state to state in what they contain, but they contain a patient information form. And this form is collected by folks like Deb, who are the sexual assault nurse examiners in our state. It contains information that assists us with our processing in the lab, and I'll talk a little bit about that as we go through this kit. So the first thing is the patient's name. We also ask for the patient's race. I've been asked about this on more than one occasion. Why do we ask for race? Later on, we're going to talk about statistics, and when we're doing statistics, if an individual is from an isolated population, such as a Native American population or an Amish population, as a couple of examples, we may need to change our statistic to account for that. The next thing we ask for is the patient's gender, male or female. We also ask transgender male to female or transgender female to male. It's important for us to know the individual's gender assigned at birth as part of making some decisions on our processing, and I'm going to show you two different workflows for our processing today that really depends upon the answer to that question. We also ask for a number of assailants, if known. We ask for each of the items collected what the suspected fluid is. Is it suspected to be semen, saliva, or a different type of fluid? We ask about oral contact. We ask about digital contact. We also ask about consensual sex within the 96 hours preceding the exam. Studies have shown that semen can remain in the vaginal vault, specifically if a cervical swab is collected, for up to 96 hours. And this is important for us to know because if there was a consensual partner, we will want a known sample from that person so we can determine if the profile we develop is from that consensual partner or the perpetrator of the assault. And this has implications later on when we're talking about entering samples into the DNA database. We will also ask about activities that the victim has engaged in. DNA remains on the body but can be impacted by the activities. So if someone showers or bathes or goes swimming or brushes their teeth, urinates, defecates, all of that can affect the elimination of any foreign DNA on the body. So that's important information for us to have as well when doing our DNA analysis. We also ask for a narrative. This is important because a lot of times victims will be much more forthcoming with you as a healthcare provider than they will with law enforcement, especially if they have had any negative interactions with law enforcement in the past. We find that we get additional information or important information from that narrative that's collected during the process of the medical exam. Our kits also include instructions for the medical provider on how to collect the kit. And I want to take a moment here and talk a little bit about that. Here in Idaho we are fortunate to have Deb Weatheralt who is our SANE SART nurse coordinator. And she really, along with training nurses to do the sexual assault nurse examinations, she also acts as a liaison between the forensic laboratory and the nurses. So she developed, along with our forensic scientists, both this information form as well as these instructions. We also have designed a feedback loop. So at the conclusion of our laboratory testing, the scientists will record, we have a spreadsheet set up, will record any issues that they noted with the kit collection. If there were issues with number of swabs, because our instructions dictate how many swabs should be collected from different body locations. And again that will be different state to state depending on how your laboratory has designed their testing. We'll tell her that if there's incomplete documentation or if there's any issues we see with the collection or how the kit was sealed. We will let Deb know that and then she will feed that back to the provider. All in an effort to have uniformity of collection and uniformity of care. Really our goal is to have equal justice regardless of where someone lives in our state. If they live in a really rural, isolated part of the state, Idaho has quite a few of those. They should have the same care as someone who's living in a larger, more urban area that has greater access to healthcare. They should still get the same evidence collection. Once I have opened the kit, I've looked at that information sheet, I'm now going to take the envelopes out of the kit. And I'm going to look at any that weren't collected and I will put those back in the box. So our kit includes a known blood sample. Ours is a finger prick and blood dropped onto a card. So I'm going to save that for last. And that's an important point that I want to talk about. Within the forensic lab when you're doing DNA analysis, there's evidence samples and there's reference samples. The testing that we're going to do and the DNA profile that you're going to see generated later doesn't tell me anything about a person. It doesn't tell me what race they are, it doesn't tell me what diseases they have, it doesn't tell me what eye color they have. The only information I can determine is if that individual is biologically male or biologically female. So it's important to have reference samples or known samples from the individuals involved in the case for comparison to the profiles we generate from the evidence. That being said, we always want to keep evidence and reference samples separate by time and space, meaning I'm going to have separate racks for my evidence and my reference. Our laboratory goes so far as to, if we receive suspect references, those are cut in a separate process and dealt with separately, and are processed completely separate from the evidence. With our sexual assault kits, the reference sample is actually in the box, so it'll be cut at the same time, but I'm going to make sure I cut it last. And this is from the person all of these swabs are collected from, so their DNA's already going to be on these samples. The suspect, the elimination samples, again, we like to keep those separate so that we're minimizing as much as possible the risk of any contamination into these samples. DNA analysis has changed a lot just in my 20 years, and a lot since its inception in the late 80s, in that it has become increasingly sensitive, and the types of testing we can do have increased. What I'm going to show you today is a typical laboratory testing, and we can talk about during the question and answer period some new types of testing that have been developed. Now that I've set my victim reference aside, I'm going to look at some of the other items in my kit. Specifically, we have vaginal swabs, external genitalia swabs, anal swabs, oral swabs, and in this kit, some neck swabs. What I'm going to do is, again, this would be filled out if this were an actual item of evidence, so I'm going to look at all of this information. I'm going to look at what's provided to me. What's important is based on that narrative, and based on what's on the envelope, is if we believe semen is present, or saliva, or possible blood, because that's going to determine some of the steps that we're going to take. I'm going to label each item with my case number, item number, and if this is item number one, I'm going to make each of these 1.1, 1.2, etc., along with the date and my initials. Once that's completed, I'm going to open the item and see what's inside. Now, because we have these uniform kit collections, I have a good idea of what should be inside, so because these are vaginal swabs, I expect there to be four swabs inside this envelope. If I can get it open here. So, I open it up, and again, I'm documenting all of my notes in here as I go, so I would be typing that I have a kit, what envelopes I have, that it was in a glue-sealed condition, what information is on the front. I'm going to be documenting how many swabs were in the kit. And the first workflow I'm going to talk about today is what we call Y-screen. The Y-screen testing is more sensitive than the traditional serology that we'll talk about next, so we have more samples going forward for DNA analysis. So, as I mentioned, ISP tests every swab in the kit, every swab in every envelope. That is not true for all laboratories. So, I have my four swabs here from our envelope, and I'm going to use a single-use razor blade, take a small cutting from each. I'm targeting about one-eighth of each swab. And I'm then going to put those cuttings into a tube. And I will label this tube with my case information and place it into a rack. I will then put this back in the envelope. I will initial and date my seal. And then I'll continue that for every single envelope in the box. Next, I'm going to add some reagents, and this would all be changed in between each sample, but for the ease of filming, I won't be doing that. And then I'm going to add some reagents to each tube using a pipette. We have specific amounts of each reagent, which is a chemical that's added to facilitate the DNA process, which the next step in the process for these samples is going to be real-time PCR, or polymerase chain reaction, and we're going to talk about that next. But first, I will add my chemicals. And I'm going to add them to each tube individually. And I'll continue on until it's been added to all of them, and then we'll move to our real-time PCR process, which involves putting these in a plate and going on to our next instrument. All right, we're now ready to do the real-time PCR for our Y-screen process. You'll probably notice I have on a different lab coat. When we're doing DNA analysis, we have one part of our laboratory where we're examining items of evidence, and then we have a completely separate room where we are going to amplify the amount of DNA in those samples. Because contamination can occur quite easily between amplified DNA and non-amplified, we have to keep this process separate, separate lab coats. Our laboratory utilizes different colors, so we never get them confused. Your laboratory may have a similar system in place, but they do have to have a separate laboratory, separate door. Nothing leaves this room once it's been amplified. This is called a real-time PCR. PCR is the polymerase chain reaction, and that is the process of amplifying DNA. In simpler terms, molecular xeroxing. We are going to target specific locations on the DNA. You're going to see those later when we go over DNA analysis or data analysis, and we're going to target those specific locations for copying. So if you had a dictionary and you only wanted to copy 25 pages out of that dictionary, that's similar to what we're doing. We're going to copy each one of those 25 pages a million-fold in order to have enough DNA to visualize. For our quantitation step, it is called real-time PCR because it's performing that amplification process and at the same time reading how much product it's able to generate, meaning that'll give you an indication of how much DNA is present there initially, because the more you have to start with, the more product you're going to generate. And this does it through cycles of heating and cooling. It'll do multiple cycles of heating and cooling. There's just a tray. We're going to put our plate of samples on the tray, shut the door, and we'll use the computer software to start it. And at the end of the process, we will know how much DNA, and specifically for this, if we have male DNA present, and if we have male or low male, or if it's inconclusive, it's going to tell us that, which will help us with our DNA process and determining which samples would be best to move forward. For our Y screen, the final step is to look at the data we generated from our real-time PCR instrument. As I mentioned, our state tests every swab, meaning every swab up front goes through that quantitation process. But then we use the data from that process to determine which samples should go forward for DNA. The results, as you can see in this screen, will give us for each sample the total amount of DNA represented by this, the total amount of male DNA represented by this column, and then a determination of if male DNA is detected. For our Y screen process, we are specifically targeting male DNA because we have our gender assigned at birth female victim and believe our perpetrator to be a gender assigned at birth male. So we're using this to determine which samples should go forward. If no male DNA is detected, that's not a sample we want to take forward. We want to take forward the ones that have male DNA. Sometimes there's a low amount of male DNA, and depending on the samples in the case, if there's a sample with a higher amount of male DNA, we will choose that one over a low male. If only low male samples exist, we will look through the case circumstances and choose the one that is most probative. We'll then go back through, we'll open up the samples that are going to move forward for DNA. We'll use the same technique of taking the razor blade and cutting. We target a total of one whole swab, so if we have four swabs in our envelope, we're going to try to take a quarter of each of those swabs. The reason we don't just sample from one is because sample collection during an exam isn't completely homogenous. It's possible one swab got a little bit more of any fluid that was present than another, so we want to sample from every single swab. Again, your lab's testing protocols may be a bit different than ours. And then we are going back to that patient information form and what the nurse indicated on the kit of whether or not semen is present, and those that have possible semen we're going to put through what's called a differential extraction, and I'll mention that when we get to the extraction portion of our DNA analysis. But this is the data that helps us determine which samples are going to move forward for that process. The other type of initial processing we can do is known as traditional serology, essentially looking for the presence of a body fluid on an item of evidence. Typically, your lab is going to be looking for the presence of blood, semen, saliva, sometimes urine and feces depending on the case circumstances. This is done on sexual assault kits in our laboratory when the Y screen isn't possible. So, for example, if the victim and perpetrator are both female or if the victim is gender assigned at birth male, then we can do some of these traditional serology tests. So, today I just want to demonstrate those tests, what they look like. So, when you see them in a report, presumptive positive for blood or presumptive positive for semen, you'll know what we're talking about. So, the first one I'll demonstrate is for semen. So, I have here a couple of swabs that are known to have semen on them. So, this would be the same process I would follow if I was testing swabs from a kit. So, the first thing I'm going to do is moisten some filter paper with water, just a small amount, and then I'm just going to take that filter paper and squeeze around the swab. That's all it takes. And then I'm going to add a reagent, and what this reagent is looking for is a substance that's found in semen, not unique to semen, but found in higher concentrations known as acid phosphatase. So, I'll take one on the filter paper that I used with the swab and the one that I did not, and I'm just going to add one drop. And this is a timed reaction, but you'll notice the one that I added the or put around the swab with semen on it is starting to turn pink in color. We let this go for approximately one minute, but the one that had the swab with semen on it is starting to turn pinkish purple, and the one that didn't is having no color reaction whatsoever. And that's what lets us know if it's possible semen is present, and we can move this sample forward for DNA analysis. And again, we time this for a minute. So, if there is no color reaction within a minute, then it's designated that that was acid phosphatase negative in our testing. The next presumptive test I wanted to demonstrate is for blood. There's different chemicals that can be used for blood for a sexual assault kit or an item of clothing collected related to a sexual assault. Typically, most labs will use either orthotolidine or phenolphthalein, also known as Castlemeyer. And I'm going, there are other chemicals available, but I'm going to demonstrate these two. So, the first one will be the orthotolidine. And again, I just took filter paper, scratched it. You can barely see a tiny red dot on there, but that will be enough for the testing. So, I am going to add one drop of the orthotolidine to the one with the sample and the one that was negative. And then I'm going to follow that with one drop of hydrogen peroxide. And you'll see a pretty immediate color change with the orthotolidine. I'm looking for a blue-green color change. Usually, if blood is present, it's pretty immediate and a really vibrant blue-green color. Again, a timed reaction, so we don't let it go all day long to see if a color change occurs. If your laboratory is using the phenolphthalein, also known as Castlemeyer, you're looking for a pink color change. And this is laboratory's preference, everybody's personal preference. My personal preference is the orthotolidine, just because it's blue-green, which is a totally different color than blood. It's also a bit more sensitive than the Castlemeyer reagent. The Castlemeyer phenolphthalein tends to be a little bit more specific, meaning it won't react with as many things that are not blood. However, what orthotolidine reacts with that isn't blood doesn't look like blood, like horseradish, for example. So, same thing, just a very small amount. Scratch on my paper. I'm going to add my phenolphthalein reagent, and then I'm going to add my hydrogen peroxide. And looking for a pink color reaction, and you can see, again, it's fairly immediate on the one that had the blood on it, small in the middle, and no reaction on the one that did not. Depending on your laboratory, they may also be able to do presumptive tests. And, depending on your laboratory, they may also be able to do presumptive tests. And, again, a presumptive test means an indication that something is present, but you haven't confirmed it. So, what we're doing here is just saying, is it possible semen's present? Is it possible blood is present? We're not confirming that that fluid is there. We do have confirmatory tests for both semen and blood. For semen, we do a microscopic examination. So, we're preparing a slide from those swabs in the kit and looking for the presence of sperm. We can show you a photo on the slide here of what sperm look like microscopically. We also, for blood, have a test that will confirm if human blood is present. There are other body fluids that can be tested for, for example, saliva. There is not a confirmatory test for saliva, so it's just an indication. I won't demonstrate that test for you here today. There's also presumptive tests for urine and feces. Again, I won't demonstrate those here for you today, but you can talk to your local laboratory to determine if they perform that type of testing. I do have a photo on the slide of what the saliva test looks like. It's kind of a long, about, takes about 30 minutes. That's why we're not demonstrating it today, as well as the urine and feces. So, these tests can become important in court, and again, if your laboratory doesn't do the white screen process or if we don't have a gender assigned at birth female, gender assigned at birth male for testing, this is the type of testing that would be conducted, which is the traditional serology. So, any positive samples, just like the Y-screen, I would take a cutting and put into a tube, and this tube would then move on for DNA extraction, which we're going to talk about next. Now we've completed the initial screening determining what samples we are going to take forward for DNA analysis. We have to perform the DNA process. DNA analysis is actually a multi-step process. DNA is found within the cells in our bodies. So, the first step, we have to break those cells open and extract the DNA out of it. We refer to this step as extraction. So, this is done through adding to those samples that we cut from our sexual assault kit, adding chemicals and applying heat, and what that can do is break those cells open. We also talked about, once we've determined if a sample, if we think it has semen or doesn't have semen. With semen, we're going to do what's called a differential extraction. What that means is we're trying to separate the sperm cells from all of the other cells. Sperm cells differ in that the membrane encapsulating them has slightly different bonds that are harder to break open. So, we can apply chemicals that will break open cells, but not the sperm cells, try to separate all of that, and then apply chemicals that will break open those sperm cells. And this process then moves to the purification step. Within our laboratory, we utilize EZ-1 robots. It's a robotic extraction. It's actually using a magnetic bead technology. DNA has a slightly negative charge, and we take advantage of that by having these beads that will attract the DNA. It allows this instrument to wash all the other cellular components away while holding on to that DNA. Your laboratory may do this manually. There are chemical types of extractions that can be performed. When I started, we did not have these robots, and everything was done manually and with chemicals. And it'll just depend on the size of your laboratory and the resources they have available. But both the manual and the robotic are perfectly acceptable ways of performing DNA extraction. So, this is what our robot looks like. The samples will go in here along with the chemicals. We then close it up, start it, and this allows the scientists to walk away, come back approximately 20 minutes later, and their samples are extracted and ready for quantitation. The quantitation, we saw that instrument a little bit earlier when we were talking about our real-time PCR for our Y screen. We're going to do the same thing with these samples, except they're purified samples that we're putting on there to determine how much DNA is present. And we can set that up again manually. The analyst can choose to set up that tray manually, or they can choose to put it on a robot. Our laboratory has a liquid handling robot. An analyst can choose, if they want to set up a plate, can choose to put this plate onto the liquid handling robot and have it make all the liquid transfers. They can also do it manually. Your laboratory may be different. They may only have a manual option, may only have a liquid handling robot. Our laboratory gives analysts the flexibility to choose. For example, if I had a full plate of samples, I would want to choose the liquid handling robot. If I just had a small number of samples that I could do faster manually, I would do those faster manually. And then once we have our plate of extracted, purified DNA, we are ready to quant, and so we'll move over to our quant instrument. We've now moved into our amplification room that we talked about earlier. I have my plate of extracted and purified DNA samples, and we're going to utilize that same instrument we used in our Y screen, our quantitation instrument that performs real-time PCR. Same process, I'm going to put my sample tray on here, and I'm going to let the instrument run. And at the end, it will tell me how much DNA I have in each sample in addition to how much male DNA I have in each sample. And that will facilitate the next step in the process, which is amplification. Again, that molecular Xeroxing, copying those 25 pages of the dictionary a million fold, and it lets me target a specific amount of DNA I want to put in so that I have enough to visualize on the instrument, as well as not have so much that I'm overblowing the system. So our next step will be amplification. The third step in our multi-step DNA analysis process is amplification. This is where we're going to perform our molecular Xeroxing. We're going to set up our plate of samples. We can, again, use our liquid handling robot that we utilized earlier, or analysts can do this by hand depending on your laboratory protocols and resources. But a series of chemicals are going to be added to the DNA samples in a 96-well plate, or depending on your lab, it could be into individual tubes. Those chemicals are targeting the specific locations, those specific pages in the dictionary that we want to amplify to look at as part of our data analysis. In order to do this amplification, we have what's known as a thermal cycler, and it's just what that sounds like. It's going to go through multiple cycles of heating and cooling, similar to our real-time PCR instrument. This one isn't going to be doing any detecting. We already used the instrument that detected how much DNA. We targeted the amount we want for our sample. So that's all prepared in this plate. We're just going to put it on our thermal cycler, close the lid. We have our prepared cycles of heating and cooling, depending on the chemistry that we are using or that your laboratory is using. They'll have their cycles set into their thermal cycler, and we're going to push start. And away it will go. And this process, depending on your lab's chemistry, can be anywhere from an hour-and-a-half to a three-hour process. Again, the analyst can put this on and go and be analyzing data or writing reports, doing other things with their day, until we're done. The final laboratory step in our DNA analysis process is putting our tray of samples onto the genetic analyzer. This is an instrument that is going to be used to analyze the DNA. It's going to be used to analyze the DNA samples. onto the genetic analyzer. This is an instrument that is going to read the DNA profile. Those different locations that we targeted are known as short tandem repeats, or STRs. And this is looking at specific sequences at each of those locations and seeing how many times that short sequence repeats at that location. We call that a variable or an allele. I'll refer to it as a variable when we're talking about data analysis, just for ease of not having to use the term allele. So this analyzer is using a camera, so we're labeled at each of the locations. And it has a camera that is going to be able to photograph the sequence as it goes through. It's going to be moving through what's called a capillary, which is a long, thin tube. We have an example of a capillary here. This is what's installed on this instrument that's utilized in our laboratory. Capillary instruments can range from a single capillary, meaning one sample is going through at a time, to a four capillary instrument. Our laboratory has one of those, to one such as this that's an eight capillary, up to even 24 capillary instruments, where 24 samples can be injected at a time. Your laboratory will choose which instrument is best for them based on their sample volume. Our laboratory has opted for an eight capillary instrument based on our sample volume. So the capillary is a long, thin tube, and different repeats will move through at different speeds. So a sample or a location that has a lot of repeats is going to move slower than one that has fewer repeats at that location. So what we need to do, and it's going to do this through a polymer matrix, which is kind of a thick, viscous liquid that it's going to take that DNA through the capillary. And as I mentioned earlier, DNA has a slightly negative charge, so we're using that feature of DNA to move the sample through the capillary. So what I need to do is take my sample tray that I've added chemicals and the detectors to, and I'm going to put it on a special tray that goes onto this instrument. And then we're going to load it on the instrument. You can see in here the capillary array. It's behind this plate, which is a heated plate, so I won't open it. And these are our reservoirs of polymer. It's in this little bag, as well as a buffer that helps it travel through. This instrument can handle two plates of samples at a time, and the software will know which plate is on at a time. And then I will close the door, and it will move the samples back into place. And everything is run through a computer software or a dashboard that will tell it what samples it has on there, how many samples for injection, and it will also capture the data that we will analyze in the experiment. So I'm going to close the door, and I'm going to close the instrument. What samples it has on there, how many samples for injection, and it will also capture the data that we will analyze to determine who may be contributing to our profiles that we took from our sexual assault kit. Now that we've completed all of the laboratory processing, starting with the screening processes that we spoke about earlier, and completed the DNA analysis and generated DNA profiles, the next step in the process is to analyze that data. It's not like TV, where it gives you the picture of the person whose DNA profile it is. It requires a trained analyst to look at the data and make some determinations and some decisions about the data. The first example I have on screen is a single-source DNA profile. What that means is it is DNA from one individual. I can tell that by looking at each of the different locations that are analyzed during our process. So looking at the screen, the first location you'll see is a letter instead of a number. That letter represents the sex-determining chromosomes. Amylogenin is what it's referred to. An X means the individual is biologically female, and an XY means that individual is biologically male. Next, we're going to look at all of the different locations that we tested for and talked about when we were going through the amplification process. The way to determine if something is single-source is to first, one way, is to look at the number of variables at each location. Each person will receive one variable from their father and one variable from their mother. In this particular profile, I'm seeing no more than two variables at each of these locations, and the locations are identified by the name across the top. So there's one location, there's another location, and so on throughout all the locations tested with our laboratory's chemistry. Your laboratory's chemistry may be a bit different. All laboratories analyze the same core 13 STR loci, and then their different kits may have different additional locations that are examined. So in looking at this profile, I can look at it and make sure that at every location I believe it to be a single-source profile, which I do. And so I will determine this is single-source and move on to my next evidence profile. Just like in the lab where we first examined the evidence samples before we looked at the reference samples, we're going to do the same thing here. It's important that the analyst not be biased by looking at any reference sample information. Our laboratory, because of our batching system that I talked about earlier, the evidence samples are analyzed before even looking at the reference samples, which are typically in a separate batch of data. So I've looked at this one. I've determined it appears to be single-source. Again, no more than two variables at any location. When I have two, the different peak heights, peaks at each variable appear to be relatively even. Again, another sign that this is from a single individual. Now, in DNA analysis, especially with sexual assault kits, we will sometimes have mixtures of more than one individual. So more than one person's DNA is present. Here's an example of a sample that has more than one individual's DNA present. As you can see, we have an X and a Y. And then looking at the different locations, I have more than two variables present. That's an indication to me that this is not a single-source sample, that I'm looking at a sample with more than one person's DNA present. At this point, it's up to the DNA analyst to make some determinations about how many individuals they think are present. In looking at this one, again, we're going to look at those heights. Do they look even? Do they not look even? So looking at this particular location, I'm going to say it looks like two people. I have four variables present. Two of them appear to be even with one another. The other two appear to be even with one another. And the analyst will go through every single location, looking at all of that information. And those are just two small pieces of information that they look at. But again, they're looking to make those determinations on those evidence profiles before they ever look at any reference profiles. Once that has been completed, and they've looked at all of the evidence from that case, the next step is to make those comparisons to the reference samples. And the reference samples will look the same as our single source profile, except we will know this one is from person A, this one is from person B, person C, etc., depending on how many individuals are involved in the case. The next step is to calculate a statistic. In DNA analysis, any time we are going to attribute a DNA profile to someone, or if we think someone is included in the mixture, it's important to provide a statistic related to that. That will let the trier of fact, which in criminal cases is the jury, make some decisions on what does that mean. If I say this DNA profile matches person A, does that mean 1 in 10 people, or does that mean 1 in 10 octillion people? And it's important for them to have that weight, to know what weight to give this evidence. If it's 1 in 100 people, they may weigh that much less than the 1 in 100 octillion people. Our laboratory uses software known as probabilistic genotyping. Not all laboratories use that, and there are different probabilistic genotyping softwares out there. Your lab may do a different type of statistical analysis, but I'll show ours for the purposes of this demonstration. So again, the first thing was the analyst needed to determine how many people were present in that mixture. We have to tell the software that. The software can't make that determination on its own. So in that mixture that we looked at, we decided there were two people, so we've told the software we believe there are two people in this mixture. There's also a feature in our software where we can tell the statistics software if we think anyone is included by nature of the sample. And what I mean by that is, if we're looking at a vaginal swab collected from a sexual assault kit, we can reasonably assume that the person that swab was collected for will be represented in that sample, that their DNA will be present because it was a swab of their body. This helps the software determine the profiles that are comprising the mixture. So in this example, and this is a proficiency test example, these are not from real cases or real individuals. So in this case, we determined that based on that, one of those individuals could be included in that sample. So I've told the software that this is a known contributor. Our laboratory calculates likelihood ratios. A lot of laboratories across the country calculate likelihood ratios. And in that, we're determining the likelihood of one scenario versus another. Usually designated hypothesis P, hypothesis prosecutor, meaning the suspect is included, and hypothesis defense, meaning the suspect is not included. And it's just weighing the likelihood of those two scenarios to determine which one is more likely. The software is going to give us some statistical information right here. I won't bore you with going through each of these statistics, but it's an indication to the DNA analyst of whether or not they were correct on the number of contributors. So it's just important information for them to look at to make sure they interpreted that mixture correctly. We'll then go down. Again, some more information for the analyst to determine if they interpreted that mixture correctly. Now it's going to give us the components of the mixture. So the contributor one is always going to be who you told it was in that mixture. That's why it's always 100% that person was in the mixture. And then this is the software's determination of who the other contributor is to that mixture. Again, this is all done before we have told the software what our suspect DNA profile was. This is all done in advance of that. And then once we've determined this, we've asked the software, who do you think is in this mixture? We will then run it again with that suspect information present. And the software will generate a statistic of how likely it is that person is present or not within that mixture. Once that has been completed and we've made a determination on whether or not that person can be included, or if the statistic tells us they are not present in that mixture, they are excluded, or if it's inconclusive. Sometimes it could be either way if that person is included or excluded. And that's more common with really large mixtures of three to four individuals. So once we've completed the data analysis, the statistics, it's now time to write that report, that report that's gonna be issued to the agency that submitted the evidence to the laboratory. And this is also the report that will be presented to the courts. And if the analyst is called upon to testify in court. This is what our reports look like. Each agency will have a slightly different format for their reports. So the reports that come from your laboratory may look much different. They all, because of accreditation, have to include certain information, including the agency's dates, the name of the analyst, et cetera. So again, this is proficiency test samples. We go through and we start off with a description of the evidence that was received. It moves down to our conclusions and interpretations, meaning what did we do and what were our interpretations based on data analysis and statistics and all of the testing performed. It also includes what we did with the evidence. Our laboratory does not maintain evidence. We return everything to the agency that submitted it to us. That may be different in your state or jurisdiction. They may hold that evidence pending any court filings. The other thing that's done at this point and is put into the report is if a profile is eligible for CODIS. CODIS is the Combined DNA Index System. It's a software program also known as the National DNA Database. It can exist on three different levels. There's a local level. So if your lab is a state or county lab, they may be the local or LDIS. It exists on a state level, which is the ESTIS. And local labs will upload to their state lab. And then the state lab will determine which profiles are eligible to go up to NDIS, the National DNA Index System. What goes into the database is actually regulated somewhat by the FBI. So for evidence profiles, those profiles we're developing from these sexual assault kits, it will have to be from the putative perpetrator, has to be related to a crime, and it cannot be a profile that we know to be from a victim. It also cannot be a profile that we know to be from an elimination. So in the case of a sexual assault, typically that would be if the victim had a consensual partner. If we develop a profile we know to be from that individual, it cannot get uploaded. There's also convicted offender samples that are uploaded, and that is set by each state. So each state will determine what offenders they wish to upload to the database. All states are at minimum all felony convictions, and that is where Idaho is. Many states also include arrestees, anyone arrested for a felony or depending on that state's statutes. Some also include misdemeanor convictions. Again, that is set by the state. It is not set by your laboratory or your laboratory's management. It is determined by the legislators in your state. So if a profile is eligible for CODIS, it will be uploaded per your state's protocols, and it will be included in the report so that the agency will know if a profile went up to CODIS. At this point, going back to the very beginning where we talked about sexual assault kit tracking, at this point our laboratory will go into our sexual assault kit tracking information. We've issued the report. We've completed the testing. We are going to document that we completed the testing and on what date. Our sexual assault kit tracking system also allows us to document if a profile was uploaded to CODIS and the date. Later on, if we get a hit in that database, so for example, if this sexual assault involved an unknown perpetrator, if we were to get a hit against another case or against a convicted offender, that would also be documented in our sexual assault kit tracking. It allows the police agency to see that. It also allows the victim to see that that happened for their kit. That is the completion of the laboratory portion. The only other step that's left is for our agency to return this to the submitting agency and for them to retain that sexual assault kit based on our state statute. Again, your state may be different. And then the analyst is completed with this case and will move on to the next case. The only time they would revisit this case would be if it goes to court. And at that point, again, this is going to vary widely from state to state, jurisdiction to jurisdiction. In Idaho, we have variations even between counties, but usually the analyst will be notified with a subpoena, just like you would be notified with a subpoena. They will set up a time with the prosecutor to review the notes and the analysis performed. And on the date specified on the subpoena, they would go to court and testify to their results. Forensic scientists will go through a process in some jurisdictions called vore dire because they are an expert witness and allowed to express their opinions. Or what that means is our interpretation of that data, what we've determined that that data means. So they'll go through a process in the court of going through all of their qualifications, what qualifies them to testify to their expert opinions. Once that's done, they will testify to their report and their results. And again, as we talked about when we were in the lab, our objective is to be objective, to not be subjective, to be analysts of the evidence, to speak for that evidence and to report our opinions accurately and express them accurately to the court system in an objective manner and to not be biased in any way. So that's what our scientists will strive to do when relaying the results of our testing to the courts. Thank you for spending part of your day with us today. Hopefully you've learned about how sexual assault kits are tested here at Idaho State Police Forensic Services, as well as at your laboratory. And we'd now like to open it up for questions if anyone has any questions. We'll also talk a little bit about some upcoming DNA testing that may be available to you. All right. Thank you. And welcome back. As mentioned, we're going to talk a little bit about some additional testing. So the other technology, and we'll go through this quickly so we have time for questions at the end. So some of the other testing that is available in many, many state laboratories, county labs, local laboratories, is called YSTR testing. We talked about YSTR testing We talked about Y-screen, where we were specifically targeting male DNA, and that's what YSTR testing is. It's targeting that Y chromosome specific to males. It's particularly useful in sexual assault evidence when you have a really low level of male DNA and a lot of female DNA that could be overwhelming. A sample where you can't see the male DNA profile, and that's what that is used for. Again, that's laboratory to laboratory. Many labs offer this type of testing. Another one that's not on the screen but that I've had questions about in the past is RapidDNA. You may have heard about their instrument that allows a person to put a sample in. It goes in the instrument, and in about 90 minutes, a DNA profile is developed. These were specifically designed for a booking station type setting where someone is arrested. A sample can be analyzed quickly to determine that person's DNA profile. As of right now, evidence profiles generated using rapid instruments are not eligible for upload into that CODIS database. So forensic labs are not using it for sexual assault kit evidence or other criminal case evidence. There is a laboratory, I believe it's the state laboratory in Kentucky, is doing a pilot study. They're taking some samples, so a few swabs, and going through their regular process, and a few swabs, and going through the rapid process to determine if it's viable for this type of testing. I've seen very limited results, so I think they are still in the process of working through that. The last one I wanted to mention, because it's one you've probably seen a lot. I know I saw a lot of Colorado, New York, other states like that, including Idaho, pop up that have genealogy programs happening in their states. And this is for those cases where we went through the whole process that you just watched. We uploaded a sample to CODIS, and we're not getting any hits. So all leads have been exhausted. That sample can then be put through this genealogy process. A different type of DNA profile is generated, specifically looking for what is common among family members. The testing we're doing, as we talked about, is looking for what's unique to an individual. And this is looking for shared parts of the DNA. And then going through family tree research and trying to determine who that individual is that may be the source of that sample in an effort to solve some of these cold cases. That was the completion of what I wanted to talk about today. So I'll turn it over to Shea and see if anybody has any questions. So James Gotti, he said he had a case come back as inconclusive. What does this mean? No DNA evidence found? Not enough DNA? So that can be a couple of things. Sometimes it can be inconclusive if it's a really large mixture. So each laboratory will set how many contributors they'll interpret. Our laboratory is a maximum of four contributors. So anything above that we'll say is inconclusive. We can't determine who's in that mixture. It could also mean that it was a very low-level profile and that no conclusions could be made on who could be a source of that because there's just not enough present. I'm not seeing any more chats coming through. So we also have Deb here. If anybody has any questions about our feedback and her incorporation into the laboratory, if anybody has questions, she would know better how that's going probably than me. So, OK, somebody asks, how long does this process take per kit? So the actual process, the laboratory process takes probably one to two days, depending on how many items are in the kit. The statistics analysis takes probably a couple of hours to more, depending on how complex the profile is. But every laboratory doesn't get just one kit at a time and processing one kit at a time. So laboratories have backlogs. And our laboratory's goal for turnaround time is to complete that initial screening process and let the agency know if they have male DNA present within 30 days of receipt to the laboratory and then another 30 days for completion of the DNA portion. That's our goal. We're striving to meet that. And then laboratories are also dealing with some laboratories with the backlog of untested sexual assault kits and working through those as well. So that can impact turnaround times. So it varies laboratory to laboratory, but the actual hands-on process, I would say, would be about a week once you do the laboratory, the statistics, writing the report, having someone review everything and getting it issued out. We've tried to focus, as you know in this webinar, to be centric to what all crime labs do. Now, we might be a little bit different than other labs or your specific lab in your state or your location, but most labs do things similarly. But check with your local lab or your state lab where you're living because they might just be a little bit different than what you're hearing on the webinar today. But again, as Raylene has very well communicated, the batching process is really what makes all the difference when you're coming to how fast things can be processed, what kind of analysts they have in the laboratory, what kind of instrumentation they have. All those factors are so important when determining how long it's going to take to work a case. And we would also, I'm going to sell out my colleagues a little bit across the country in that most of us are very willing to host tours of our laboratory facility. If you reach out to the laboratory management, especially when there's a purpose with a stakeholder or a participant in the process such as the nurses are in this process, to be able to educate and to be able to build those relationships with your state and local labs, it's really important that you are in there talking to them, interacting with them. We've made that a natural nexus with Deb's position in integrating the nurses into our system, and that doesn't exist everywhere, but it can exist everywhere if we all extend ourselves a little bit and be open to those communications and those dialogues. We'll also offer up our assistance there that if anyone does need assistance contacting their local CRAN lab, they don't know who to contact, please send Ryleen or myself an email. We will find the contact for your laboratory so that we can make those connections happen. And I will share my screen here again quickly that has our contact information on it. So there's all of our contact information, and like Matthew said, we're always ready to answer questions or put you into contact with someone in your state that can help with the specifics for your state. Thank you. And just for everyone's knowledge, Matthew's on our group of partners for our project, so we can also put you in touch with him if you reach out to us. Absolutely. And we're so proud to be a partner with you. I'm so proud to be a part of this collaboration to make things better in this community, and we just value all of those partnerships that we have with all of you. Thank you. Thank you all for providing this for us. So I think we can close out now.

Crime Lab Analysis

Sexual Assault Evidence Kit

Arrival at Crime Lab

CODIS Entry

Ryleen Nolan

Idaho State Police Forensic Services Laboratory

DNA Testing

YSTR Testing

Genealogy Testing

Collaboration

Laboratory Accreditation