Wisconsin Public Television University Place Wednesday Nite At The Lab  "Innovations for Screening Newborns"  November 3, 2010 UP515    cc >> Tom Zinnen: Welcome everybody to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the University of Wisconsin-Madison Biotechnology Center. I also work for UW-Extension Cooperative Extension. And on behalf of our partner, Wisconsin Public Television and the Wisconsin Alumni Association, welcome to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, I'm delighted to introduce two people from the Wisconsin State Laboratory of Hygiene, which is approximately 48.3 yards up the street here on Henry Mall. I measured. The first director or the first person is the director Chuck Brokropp who grew up in Lancaster, Wisconsin. Went to school at the University of Wisconsin-Platteville where some other major famous people went. 

And thank you. Alright, maybe some minor not so famous people. Oh god no, do I. (LAUGHTER) And as a doctor of public health as you see from DRPH which is the first question tonight already. And his colleague is Mei Baker who got her training in medicine and research in China and has done research in medicine and public health here in the United States. And they're going to be talking about SCID, the severe combined immune-deficiency disease of newborn children and the new way they have figured out how to test that in newborns that I think is going to go not only all the way across Wisconsin but soon will be used across the nation. I don't know which one of you is going to go first. Please join me in welcoming Doctor Brokropp, Wednesday Nite at the Lab. (APPLAUSE) >> Thanks, Tom. It's been a couple years since I had a chance to talk to Wednesday Nite at the Lab. 

It's always a time to kind of share some interesting things that have been going on. 48 feet or did you say yards that way? >> Meters. (LAUGHTER) >> Too scientific for me, I can't handle that. But tonight, Doctor Baker and I would like to share with you and tell you a little bit about our lab and share with you a really exciting research project and public health project that we've undertaken here in Wisconsin. First of all, I'd just like to briefly talk a little bit about the state lab of hygiene. It's Wisconsin Public Health Environmental Laboratory. It was formed in 1903 and is one of the leading public health laboratories in the country. We have two facilities, one just up the street a ways and another one out on Agg Drive on the East side of Madison. The state lab of hygiene is specifically attached to the University of Wisconsin-Madison to take advantage of the research capabilities and the teaching capabilities to promote public health here in Wisconsin and more recently across other states as well. What do we do? Basically four things, real simple. 

We do reference testing on clinical specimens, we do testing for, on environmental specimens, on clinical toxicology specimens, on forensic tox specimens. We're involved in teaching. A number of our staff have faculty positions here at the University of Wisconsin. We have a pretty extensive research mission that I'd love just to stand here for a couple hours and go through all the projects that we work on but Dr. Baker and I have just chosen one for tonight. Maybe you'll invite us back. We also have a research mission where we reach out and work with clinical labs and environmental labs throughout the state by providing them with training and other types of laboratory services. One of the programs at the state lab of hygiene is newborn screening. Every time a baby is born in Wisconsin, one of the things that usually happens to that baby is that some main nurse comes along, picks the baby up, squeezes the heel, pricks him in the heel, and collects five drops of blood. Now that doesn't sound like a whole lot because they collect it on a piece of filter paper. 

That filter paper with the blood on it is sent into the lab and we test that for a whole host of congenital and metabolic conditions. This is Doctor Bob Guthrie who in the mid '60's came up with the, he identified the cause of a fairly common condition known as a PKU or Phenylketonuria. If that condition is not identified early in infants, and treated with an appropriate diet that infant will develop some very severe retardation, it will not develop normally. And in the past, before we started screening all infants for this, these infants would be so affected that they would end up in like state hospitals. Central Colony here, just on the other side of Lake Mendota, used to have hundreds of these infants at any one point in time. But since the '60's, when we began, we developed a test that we could screen for these infants. We no longer see infants that are that affected because once we identify them, they can be put on a special diet and basically go on to live a pretty much normal, normal life. >> Don't they outgrow it? >> No, they do not. I mean, this is a metabolic condition that pretty much is something that involves, that they have to deal with most of their life. Here in Wisconsin, we're one of the states who now tests for 47 different conditions. And these are just some of the ones that we test for. And we do that all off of that little spot of blood that's placed on the filter paper. 

We basically take a little punch of that, put it in a tube, and then start with that dried blood on a piece of filter paper and elude it and run all these various tests for the conditions that are listed on this slide. I highlighted the one we're going to talk about tonight and that's Severe Combined Immunodeficiency. This is also known as Bubble Boy Disease. Several years ago, John Travolta was in a movie and there was an ad. The movie basically portrayed a young child by the name of David Vetter who had this condition, and basically lived for 12 years inside this plastic bubble. The reason he was living in the bubble was he did not have any viable, functional immune system that would protect him from becoming infected with a bacterial or a viral illness so in order to live a relatively normal life, he had to stay within the bubble. Now, Severe Combined Immunodeficiency is basically a life-threatening condition. If it's not detected early in life, the child will be diagnosed usually at about 3 months of age, they'll start coming down with one infection, might treat that, then they come down with another one, then they come down with two at a time. Then they never get over one and usually by the time the child is 12 to 24 months old, they will succumb to these infectious diseases. So it really is a serious condition in children. It's basically the result of normal immune cells simply are not present. 

They don't develop normally prior to birth or shortly after birth and, therefore, they cannot produce the immune response that is necessary to protect them. This is important because today, infants and young children are often given live viral vaccines and if you give one of these vaccines to one of these infants who doesn't have an immune system, instead of just producing an antibody or a response to it, they will likely come down with the disease itself. So it's very important that we identify these children and not subject to some of these vaccines. The other thing that's important with this disease today is that we now have a way to cure these infants once we identify them early in life. And so this is really one of the first times where we've been able to apply today's technology that Doctor Baker will talk about, something called PCR testing, it's a molecular test and apply that in newborn screening and as a result of that, there's a treatment that can actually cure the infant who is afflicted with this condition. A little history on this goes back about four years in late 2006. In 2006, the Jeffrey Modell Foundation donated $250,000. The Children's Hospital of Wisconsin matched the donation. The State Lab of Hygiene put up a similar amount of money. We pulled the resources and during 2007 and 2008, Doctor Baker was instrumental in working with colleagues from the Medical College of Wisconsin to develop a rather novel assay to identify these infants who may be affected. 

In January 1 of 2008, we actually started screening all newborns here in Wisconsin for this condition. So we've been at it here for, what is it, 2, pushing 3 years right now and we screen about 70,000 infants a year. Well SCID was one of those conditions that really met the model for a disease that can benefit by newborn screening. And these are some of the criteria we would look at. You know we knew something about how common the disease is. When we went into this, there were published studies saying it was like 1 in 66,000 and maybe 1 in 100,000 births would have this condition. The disorder has a short asymptomatic period after birth so a baby could be born could have the condition but will appear perfectly normal. So without doing a screening test, he'd have no way of knowing whether the child had this condition or not. It does cost very serious medical complications and in fact, like I mentioned earlier, the children who are not diagnosed with this will most likely be dead within the first year or first two years of life. Now we do have a potential, a very successful treatment for it today, we have confirmatory tests that are available. 

And if we do an intervention early, it really does lead to better outcome, like a normal, functioning child with a healthy immune system. And is there a screening test? And the answer to that is yes. Primarily because of the work, some work was done elsewhere and then Dr. Baker was able to modify that test so we could adapt it to newborn screening and also automate it so we can screen, you know, 300, 600 to a thousand babies a day. And using this newer technology, you know it's one thing to do 2 or 3 tests or maybe 10 or 11 but when you start doing 100, 600, and more tests a day, it gets a little more complex and the automation equipment becomes much more involved. We basically added SCID to our newborn screening panel that I showed you. But before we did that, we reviewed the clinical and scientific justification for doing this. We obtained input from experts. We reviewed the need for a statutory or a regulatory change. Conducted a pilot study, we considered conducting a larger pilot study. And then we had to provide some education to the clinicians who would be seeing these infants if we were to identify them. 

And then also to develop and validate the screening method. All that work was done prior to January 1 of 2008. The one thing we had left to do was to really get the Department of Health to promulgate sort of an emergency rule to add that to the test battery. One of the other things we looked at that we thought was quite substantial and what actually convinced the Secretary of the Department of Health to add this test to the newborn screening battery was some limited data on a few number of cases from here in Wisconsin. We had information on five babies who were ultimately diagnosed with SCID. And those five babies, and only three of them survived and were still alive at that time that we collected this data, they had an average hospital bill of 2.2 million dollars a piece. That's phenomenal these days. In addition we had a child who was actually diagnosed early primarily because there was a sibling in the family that had the condition so we suspected that this next baby might have the condition. And because that child was diagnosed early and treated, the hospital bill ran only about $250,000. I say only. 

That's still an awful lot but you can see it's almost you know only a tenth of what it costs if we're not doing the screening. To take those numbers and compare them to what it costs us to screen seventy thousand infants in Wisconsin a year, at about 5 dollars a piece, you know we're only spending about $350,000 to screen every baby for it and when we do that we are starting to pick up some of these infants with Severe Combined Immunodeficiency and other immunodeficiencies that Dr. Baker will mention in just a few moments. We took this information to the Secretary of the Department of Health and to the Administrator of Public Health Department and my predecessor, a good friend by the name of Dr. Ron Lusig, was pretty, he had retired but yet there was one more thing he wanted to do. And that one thing was to really see newborn screening expand by adding screening for SCID. And so he was pretty persistent in the way he went about making sure this would happen. And these three individuals put their heads together. You know, after they were done discussing this, they looked at each other and they said, "Why shouldn't we do this?" And there was no reason why we shouldn't add this to the newborn screening battery here in Wisconsin so instead of going through the normal rule process that may drag out for months to several months, they basically moved forward with an emergency rule because every baby we missed was going to basically have a poor outcome. And so on January 1 of 2008 we began screening all infants here in Wisconsin. Well that sort of set the precedent for the rest of the nation and since that time the experience that we've had here in Wisconsin has picked up to where the Secretary of Health and Human Services has basically approved a recommendation that this particular screening should be added to newborn screening programs in all states. And now that that's moving forward, we're beginning to see requests from many states. 

I think there's 15 or more states now that have requested our assistance in helping them bring the test online and sharing the information that we've gathered here in Wisconsin, how to go about adding this to our panel. The National Institute of Health has recently funded 22 million dollars to bring other states online. A lot of that money is going to California where they hope to be screening at least a half million of the 650,000 births a year in California. The state of Massachusetts, New York, Texas possibly will be coming online, and just recently, we began screening all babies born in the state of Louisiana. The blood samples are taken in Louisiana, they are punched into a little plate, the plate is sealed, they overnight them to us. We bring them into the lab and just slip them in and run them right along with all the babies here in Wisconsin. So this is something that's going to catch on and move quickly across to the other states and also internationally.  (INAUDIBLE) Yes and Dr. Baker will mention that, she has that on a slide. And that's an important thing because when we went into this, we didn't know exactly what it was. But what we're finding out is the test not only picks up SCID, the traditional condition, but we also pick up other clinically relevant immunodeficiencies so it actually has more broader application than what we had thought. 

I think I'm going to quit with this and let Dr. Baker tell you what's really important about all this stuff.  (APPLAUSE) >> Mei Baker: Thank you. I hope that Dr. Brokropp convinced you that this is a very serious disease. We detect it early and we are doing whatever we can trying to save lives and I'm going to take you back a little bit to describe how we use the test to do this screening disease. And in the newborn screening setting, we want to detect disorders, this marker has to be fitting certain criteria. And the marker we choose to do we call a T-cell receptor insertion circles. I want to talk in more detail. And the reason we choose to do that is a DNA fragment in the --  body is a circle DNA fragment and the one I show you in the picture will make more sense. Also they are important in a functioning way is their form during the T-cell maturation. So this is a very, a good marker is a function marker. 

And these days we talk about DNA a lot, talk about DNA, talk about genetics but its beauty here is we use a DNA marker to link to the function so this will make more and more clinical utility. So, we'll talk more about that. And also this fragment, actually after maturation, T-cells, when cells go through more division and they're not reproducing themselves, so this is a perfect marker at newborns because just all things are in the active stage and it kind of has a --  unique situation. Coming to this is not just out of the blue, we thought about it and we did the research and when we choose the marker, the person listed here, Jennifer Parker, she's in the UCSF, and she was the first one to think about that with the previous experience by other people in the field using this marker to evaluate the treatment actually in an HIV infection. Because HIV is - SCID is a primary immunodeficiency - HIV is acquired because it's a virus infection and destroying in the system. When you have treatment, you want to know how your immune system is reestablished so which is a marker and which is -- used as a marker so Dr. Jennifer Parker is very smart enough. This, again is a function marker and in the -- immune system so why we cannot use this to apply in the dry blood spots for the newborn screening and to indicate the disease. And the base of Dr. Jennifer Parker's work and we did further because, again, Dr. Brokropp indicated in the laboratory, you do 10, you do 20, everybody can do that. Can we apply this technology back to the Public Health laboratory and we can deal with hundreds or thousands and this is another very challenging. And we were further able to refine the tests and develop a high throughput in the test so we can accommodate hundreds or thousands who come every day, every week, every month. 

So our experience has to generate a certain publications so we want everybody to know, more people can learn, the more people can do. Our medical paper published in the General Allergy and Clinical Immunology. Our first year screening experience has been published in the General American Medical Association and I think this is a paper everyone knows and also in our experience, how to bring a new disorder, take into the current setting and integrate it with other forty six different disorders and this experience we have reported in the Public Health Report. Now we go back and think about the T-cell and hopefully this little cartoon can help me to explain it here. First we start we call the lymphocytes --, it's in a very very early stage and that is a stage that don't really have function, and needs to get more education, and premature, and usually in the fetus or liver. When we go in, we have a In the body, the immune defense have two arms, one is T-cell, more dealing with virus, this kind of thing and the other is B-cell, more dealing with bacteria. So how that works and how that becomes function, first they are going to the bone marrow in the B-cell and gain full maturation so they can generate antibodies. And the T-cell is different. The T-cell is going to the thymus which is a very, very small organ. It's about this big, like my thumb, and in that thymus can be perfectly described as a school house. 

And all the cells go in there, the T-cell goes in there, and get an education. This is an education I would describe not as a college because you can't just choose what you want to do, you have to do sequential. You start with elementary level and then go on so we can start here, then you can go to middle school, then you go to high school, and also all the cells you educate at a different level. So your cells become smarter and smarter and generate a certain function to begin with and also I want to mention to you is our body is designed amazingly because in the environment we have so many, many different antigens. And our body cannot make all of them. The way they want to do more, the more efficient way is we know DNA is a blue-copy of our life and we have a copy. But the copy that going through some change, so generating a lot of variety and trying to deal with so many diversity and antigens. So the way that the body did is those T-cells surfaces have a receptor to recognize different antigens. And the DNA decoding this and going through a recombination. And the recombination generate different diversity so during  the process, this is not a random, this all have the genes regulated. 

So here on the bottom, you have listed different genes. In the different stages, you have a different gene to control them. Can you imagine logically if here the gene defect will block this process and any stage, no matter in which stage, any stage that you come to the T-cell deficient because they cannot further development. And during this development stage, the DNA recombination and you generate a small circle DNA and that we call a T-cell receptor insertion circle. And can you imagine if you have this development blocked and don't go further than this circle DNA will not have formed. So in the end, you have much, much less. So that's the way we use this marker. Undetectable and lower will indicate you have some T-cell development problem. And next slide will talk more about the T-cell receptor insertion circle we call a tracks. The tracks we used here, a couple here to help me to demonstrate, you know you mentioned DNA is like a rope-like thread here, a rope here. 

And this piece like the first one here and DNA going through the recombination process, we have a piece DNA, we call excised, going this circle. And this part will come out from this strand and then you have two pieces now. So this is the small piece. When it comes out, it comes in a circle, we call the T-cell receptor insertion circle. And now you have another fragment. So that's the way how that happened. And can you imagine here now is first we call this DNA come out from journal so it's not in a chromosome anymore. And this becomes a circle and usually is more stable so that if your cells in the body for quite a long time and also because not in a chromosome anymore. When cells go through division, one become two, two become four, four become eight, and beyond. And their not a regular anymore because they're out of control, out of --. So over time, that can become diluted. It's all relevant when you use a clinic to use a marker for the test. 

So again kind of just a summary what an important point we would want to make. First, we know the principle comes from, T-cells, TREC's are zero would be our disease stage. And also we use DNA. Because the DNA we use promotes a chain reaction to copy whatever stable copy and increase them to whatever we analyze that. The reason is when you become a circle DNA, we call a primer, measure the two and do many, many cycles so that the 1 becomes 2, 2 become 4. But usually increase the copy numbers to whatever analysis. So this we call a PCR. And in our process of course we want to, we don't want to use this until we feel comfortable and confident. So we use -- and -- samples to make the assay good and also all things under and approved by the IRB. This is just showing the process by how we do that. 

Here, Dr. Brokropp showed you, we come in with dried blood spot cards. From this, one of five spots you punch a small punch. The punch is three millimeter size and the equivalent of three microlitre of blood is very, very small amount, and in the ninety six well plate and we will do the DNA traction. After that we do a real time PCR and generate a lot of copies. And then this is what the machine looks like. And of course everything is computerized. See the data on the computer and here is the picture and in order to find the copy number, we have a calibrate at a low concentration and seriously diluted. So this is how all the samples must be tested and according to the standard curve, we know the copy number. This is a kind of bigger picture to show you. Another way to demonstrate it here is the different concentrations of calibrate and here is a blue number we want to test and see how many copy numbers are there. 

And again this is a screening test. And a quick, defined way what's the high risk for this disorder group. And for newborn screening, you always want to have a confirmatory test to quickly tell you what the screening you identified a group of kids really is truly a disease. Because you started with a big seventy thousand and you really want to limit it to a small number so we can take another look and more details. And for these disorders, we call a flow cytometry that require a couple CC liquid blood and use more advanced equipment. And to we call the T-cell subpopulation, because we talk about the T-cell deficient and T-cell is a part of the lymphocyte. So when we got that, we want to know if truly the B-cell or T-cell uses that marker. Here is what the marker looks like: CD3, CD4, and CD8 is the T-cell marker, and CD19 is the B-cell marker. So we will further confirm is if these kids truly have T-cell deficiencies. And here is so far we screened about 200,000 babies in Wisconsin. 

And a couple things you can notice here is you know we separate full-term and pre-term. And Wisconsin's birth date, we pretty much see 10% of premature infant. And premature infant, their gestation is anywhere between 23 weeks to 36 weeks so the development stage is behind full-term. So we tend to see a lower copy number, seems logical, makes sense, so we treat them a little bit different. And for other disorders they have --, so in Wisconsin, we have the program in place for premature babies. We do screening tests more than once and try to very hard to more accurate, also trying to avoid any false negative or false positive. So that is a standard and the NICU all understand that. So, when premature babies have a first screening and every two weeks they will send another one, every two weeks another one until they discharge. So, SCID is not any different than other disorders. So when we see premature babies have a lower number, and we tracking, make sure until we know, and safe to say okay, and only full-term babies will we take action immediately. 

So this is an important concept because, for the newborn screening, we do 70,000 babies and we really, really want to do our hardest not to generate any anxiety for the parents. So we want to keep very, very small numbers. We know we have confidence, we need to take another look so we will not miss any disorders. So in this numbers you can tell for this 200,000 babies, we only have like 100 families who either want to do the --, confirm a test like I told you before. Also, we have another situation because this test is based on the DNA and we want to make sure the sample really gives us good DNA to study with. When you have no good quality DNA, we said, you know we really don't want to give any conclusion based on this not so ideal sample. So in this situation, we said let's repeat, do another look and instead of going to confirm the test which is a set of money, because we do another spot, it's much, much simpler. So on this and I can tell you and probably tell you, this assay it's perhaps the best screening assay in our newborn screening setting and we look at that .05 screening positive. When Jennifer Park introduced this idea, her publication, does screening positive rate is 1.4 and ours is .05. So, the practice, the fact and really give us more confidence that yes, we did it right. 

This is a good way to do it. And so screening this many babies, what do we see? Our target is SCID. SCID is one of primary immunodeficient. How many we see so far, people are anxious to know. And I can tell you in this cohort, I can tell you we have identified five severe cases. When I say five severe cases, these kids need bone marrow transplant. Without that, they would die. I can tell you a couple cases, if not newborn screening, that couple babies would not be here today. And the five cases, we have one case, this one here, Rac2 mutation is the second case in the whole world. And this baby is beyond T-cell deficient. 

Besides T-cell deficiency, it has a neutrophyl deficiency and this kid in our immunology described it, combined combined immunodeficiency. This kid would have died and nobody has question about that and because the bone marrow transplant, this kid is doing wonderfully. And the mom brought the baby, toddler already, and come to our laboratory and visit. It's quite a good experience. And also we have actually in our institution, we have a SCID baby and literally zero T-cells and because of newborn screening, we found it and the kid was protected right away and we prepared a bone marrow transplant. And which happened and doing wonderfully. We already see the bone marrow have new T-cell generated. And we have another case, and just one through the bone marrow transplant and still you know need a more longer time for observation. And one case we prepared to do the gene therapy through NIH. And besides these things, we see other, remember again, I said this is a function marker and so you can pick up other deficiencies. 

For example, DiGeorge, DiGeorge is a chromosome disorder and 22q11 deletion and this disorder has one of the things is they have a thymus degenerate. So think about that. You don't have school, where'd you get an education? You don't. And then you don't have a good T-cell function. So we pick up this and it is not surprise. Also, we have seen some congenital abnormalities. Especially if you have a -- fluid leakage, and get out of your circulation and it's not surprise when you test the blood, you don't see much there. So we see that and after surgery repair and things resolved. So this kind of thing, we see this. Just give a sense, really is a function marker, really when it's done then have ---. 

And part of what we do we want to save a life and do good things for Wisconsin. Also we want our larger -- to assimilate to other people that can do and we should, I mention this, a citation here if you want to read it more. And another one. And in conclusion, our experience really indicated first, newborn screening tracking has allowed for a high through put and to the population base of screening. And the part we had in the laboratory, can do it. And the second, the assay-- Because it probably helps and we want to make sure it is cost effective and cost efficient and as Dr. Brokropp said, it is about $5, and if you go check, especially down in the lab, how much it costs any more than our test, and this $5 you cannot do anywhere. So we are very pleased we can do that. And so it is feasible in the laboratory. And I think I can convince you and we can pick up SCID and other primary immunodeficiency. And because the bone marrow transplant and early detection have really saved lives. 

>> For the bone marrow transplant, where do you get the bone marrow? >> Good question. And now we have the nationwide registration, the bone marrow transplant is more really of a concept and the technology developed you can use a quart of blood. And all the transfusions is actually just like an IV. And the stem cells know how to find a home. They will find a bone marrow, will stay there and they will reconstitute. Well, you know, we have done this and I think it seems going well but we want to go back, think about how this thing started. Like Dr. Brokropp said, you know, we really appreciate the funding research so we can do this work and the Jeffrey Modell Foundation have the Chancellor Fund, $250,000 started and the Children's Hospital will match it, and the State Laboratory of Hygiene have put a lot in that, both financial and the site, and all this thing, and also we are very pleased with our first year success experience. We were able to secure further funding from CDC, $500,000 per year for three years. So we are on into the third year. Also, and I really think everything happened and not any individual can do this such a thing, it takes a village. 

And the list of people I don't really have time to read through details but I am very, very pleased to be part of team. Thank you very much for your attention. (applause)