Going out with a bang!

I can't believe its already the very last day working in the labs. It'll be sad to leave the lab but I'm excited for the poster session tomorrow!
Today was transplant day for one of the two patients waiting right now--very exciting!!
I was able to observe in the operating room with Dr. Markert, Julie (who works in the lab) and two other students (Dave and Ashley). After we carried over all the slices of thymus in a plastic bin and suited up in bunny-suits, hair and shoe covers, and masks we entered the operating room. A resident was actually performing the surgery with one or two doctors and several nurses, and us. It was a pretty full house but luckily we were in a pretty big room. They had started to put the central line into the baby when we arrived and it took them a while to get it positioned correctly. They start by putting a needle near the neck and then using it to guide a wire through a vein all the way to the heart, and then thread a tube over the wire and pull the wire out. They used x-rays to watch the position on the wire which we could watch on a screen. The central line will be used to give the patient calcium and nutrients while he's waiting for stomach surgery. They then started the transplant procedure by making an incision on both thigh muscles. Fortunately, one of the students was able to recognize at this point that they were not meant to be a surgeon and had to go sit down right away. I went out into the hall when the person fainted. Luckily, we were already in a hospital and some nice doctor brought us a wheelchair and some food. And so...i missed most of the actual transplant surgery. I was able to go back in the operating room just in time to see the last thymus slice inserted and watched them sew it up. Needless to say, it was a very interesting day at the hospital all around and a high light of the summer!
I'm so grateful for this opportunity, i can't imagine a better way to have spent time working this summer. Everyone in the lab made it such an enjoyable and amazing experience. Thanks to everyone in the group too, it's been fun!

Last week was a pretty slow week with most of the group on vacation. I continued to watch the jurkat cells and count them on a pretty regular basis. This week there should be enough cells to harvest them and extract RNA. I also observed the extracting of PBMC's (peripheral blood mononuclear cells) which contain the lymphocytes and monocytes (cells of the immune system). When spun at high speeds the blood separates into different levels according to density. The lymphocytes and monocytes form a buffy coat under the layer of plasma that you pipette out. It's important to get good separation of the blood for clear results in many of the tests in the lab. Once the pbmc's were separated Jie showed me how they select the T cells (a type of lymphocyte) from the sample magnetically. T cells express CD3 on the cell surface. The white blood cells are mixed with CD3 antibodies that are attached to an iron molecule. These micro beads only attach to the cells expressing CD3 and the whole sample is run through a filter with a magnet. It was cool to see the process from beginning to end starting with whole blood and ending with a specific type of lymphocyte.

It also looks like I've got the rest of the data I need for my project. It's exciting to see the last bits come together. So far our data suggests that the CD56hi cells we were looking for are present pre-transplant in babies with no thymus function. This does not support the hypothesis in another paper, based on mice, which proposes that this type of cell is derived from the thymus.

Babysitting Jurkat Cells...

Last week was another pretty productive week in the lab. My data study is coming along. A couple of key points are still missing (they're somewhere in our records, but hard to find since they go back to 2006). I also put together data on adult control samples of blood to figure out what a "normal" range is for Natural Killer cells (a type of white blood cell). This is pretty useful since the same adult volunteer can have a big range in NK cells over time, and when you're comparing them to patients it can be misleading.
I also started doing more work in the lab this week. My job was to recover a human T cell line called Jurkat cells. The Jurkat cells needed a specific medium which I put together. Then came "waking them up" which involved finding the right tube in the liquid nitrogen, and heating them quickly in a water bath. The cells are very touchy so you have to be careful and move quickly. I counted the alive cells using a hemocytometer. Every day I checked the cells under a microscope to make sure they were happy and growing. There were a lot of dead cells floating around in the flask but hopefully they don't affect the living ones.

Later in the week Jie, the same person helping me with the Jurkat cells, gave me a lesson in immunohistochemistry. IHC is just a way to see where different proteins exist in a tissue using antibodies. It was really interesting to see the process, it looked so hard! Each of the slides had different antibodies that would stain the proteins in the tissue in multiple steps. The next day I got to see the slides under a microscope. I was surprised to learn that when a patient gets a thymus transplant the tissue is not put in its natural place, above the heart, but around the thigh where there's more tissue for it to attach to. Under the microscope you could tell the muscle cells from the thymus cells by the shape and by a stain which only colored cells with a thymus markers. We were looking for cells with CMV, a virus, which was almost like finding a needle in a haystack. Looking at control slides of thymus tissue pre-transplantation was also really cool (the pictures above are of normal thymus tissue). Another good week...I can't believe we're almost half way done!

Hey guys, Allie here. This week started off with my first lab meeting on Monday. It was really interesting with everyone there talking about their own progress and also discussing how to answer some new questions in the lab. For most of the week I went on entering data from flow charts on patient's NK cells. Several of the records went back to 2006 so the data had to be re-gated for my specific study. Jen taught me how to use a program called FlowJo, which lets you interpret the data straight from the flow cytometry lab, which was fun.
When I wasn't working on data analysis Dr. Devlin and Dr. Markert spent time teaching me about other important concepts in the lab. Patients with atypical DiGeorge are born with some T cells, but because the babies are athymic the T cells can't recognize self from non-self. These T cells are likely to bind with self antigens and proliferate. The result is large oligoclonal populations and a terrible rash for the patient. Spectratyping is a tool that uses RNA to show you the diversity of the T cell receptors. An atypical patient might show a large T cell population but with spectratyping you'd find they had only a few oligoclonal populations, as opposed to a normal very diverse population.
On Friday, David, the undergrad, brought his samples to the DNA sequencing lab so I tagged along. Instead of just dropping off the tubes with the lab, we got to sequence it ourselves which involved combining the DNA with a mix of primers, enzymes and nucleotides (some fluorescent) and loading the samples into the machine, the very very expensive machine. The actual process was a little anticlimactic but learning about the how the process and machinery all worked was interesting. All in all it was another good week. Soon I'll get to do more bench work along with the continuing data analysis, so I'm looking forward to that. Hope everyone is having a great weekend!

Week One in Dr. Markert's Lab

Hello everyone! My name's Allie Stashko and I'll be a senior next year at Chapel Hill High. This summer I'm working with Dr. Markert in the Thymus Transplantation Lab. The thymus is where your T-cells in your blood come from. Babies with complete DiGeorge Syndrome are born with no thymus, and thus have no way to fight off infection. This is the only lab that does this transplant surgery, so the patients come from all across the world. Patients who have had a successful transplant continue to send back blood samples periodically to make sure everything is in check and for research.

Most of the procedures in the lab involve human blood and tissue, so I'm not allowed to handle any of it. Instead this summer I'll be doing a lot of data analysis and going back through old patient records for studies. For the first couple of days Dr. Markert spent time teaching an undergrad in the lab and me how to read and interpret flow charts. Flow cytometry is basically a way of finding out and quantifying the different types of cells in a sample. I learned some of the different assays they use to check on the levels of lymphocites and the surface proteins and antibodies that go with each type of cell. At first it was a lot to keep track of and I was constantly going back to my notes but it's getting easier and easier to recognize the types of cells by the antibodies they go with.

Today was the first day that I was able to start my project. The project involves going through old patients' flow charts to see if CD56+hi (a type of NK cell) exist pre transplant in patients and how the levels compare to post transplant and in control blood samples. Although this involves a lot of excel and photocopying, it's still very interesting and helps me to get a better understanding of what all the tiny little dots on a flow chart really mean.