Friday, August 20, 2010
A visit to Robo Lab was a great way to start the week. As someone who is interested in high-throughput operations, this lab offered a glimpse into the way hundreds of thousands of samples can be tracked and processed every day. Although the system works fairly well, there are still major issues that need to be addressed, and the efficiency should go up significantly with renovations and upgrades scheduled for next year. Some of the problems with the current system were obvious (e.g. physically moving samples across long distances rather than drawing multiple samples from tubes in a single location), while other problems were more difficult to spot (e.g. using mechanical tracks that are prone to malfunctioning or contain parts that readily wear out, and using systems from multiple suppliers/manufacturers so cohesiveness becomes an issue and technical support can be difficult to receive). It was also interesting to see the way the samples are transported through the hospital via a vacuum tube system. It would be nice to see the updated and condensed Robo Lab a few years from now.
Before selecting the genes of interest for my qPCR, I spoke with a researcher in the lab who has done studies on wound healing in mouse lung and liver samples. Based on our conversation, nearly a dozen genes were identified for analysis. The first step in ordering primers is to consult the National Center for Biotechnology Information (NCBI) database in order to identify the loci of genes as well as to find the sequence. The Harvard Primer Bank is then used to identify primers that others have reported as being useful when amplifying the gene of interest. Since others in the lab will likely use these stocks of primers for cell cultures containing both human and mouse cells, it is important to use the Basic Local Alignment Search Tool (BLAST) to verify that both the forward and reverse primer for each gene is a 100% match for the mouse gene and at least one of the two primers is not a match for the human gene. Once this is done, the primer can be ordered. Once the primers arrived, the stocks were diluted to a standard concentration, the concentration of the RNA was analyzed, cDNA was made from the RNA, and the qPCR was run. Eleven genes were analyzed along with one house-keeping gene for normalization of the results.
A preliminary review of the data has revealed which genes were up regulated and which were down regulated as a result of 45 hours in the bioreactor, and a third tissue set, involving a different media, has provided even more interesting results. Unfortunately, the company hold the components of the media as a trade secret, so it will be difficult to form a strong hypotheses as to why the change in media resulted in such a dramatic change in genetic regulation.
Summer Immersion has been a wonderful experience that has helped shape my outlook on biomedical engineering. Thank you to everyone who has helped make this possible. I look forward to hearing about the experiences next year's class will have during immersion.
In addition to lab work Tuesday I was able to attend medicine grand rounds in which Dr. Muthukumar presented a lecture on "Why Organs Fail? Molecular mechanisms of Fibrosis". His discussion of cellular differentiation complemented what I had been learning in the lab, so it was well worth skipping lunch in order to attend the lecture. Much of the rest of the day was spent reading papers.
On Wednesday I was able to make histology slides from the frozen lung tissue, and I learned that it is best to inject the lungs prior to freezing to ensure they do not fall apart or shred during slicing. Unfortunately, everyone I had spoken to had omitted this step when describing the protocol, so I was left with samples that provided a bit of a challenge. Thankfully, although the process became more time consuming, good sections appear to have been produced, and any slides I make in the future will likely seem easy by comparison. This is probably the best way to learn.
Wednesday evening I was able to attend a second MRI class in which we learned how to scan the head and abdomen. It was very interesting to see the limits of the "breath hold" technique, and I now have a greater appreciation for the work done by programmers in order to reduce motion artifacts. It was nice to learn more about the fundamentals of MRI (T1 vs T2, how to get Susceptibility SPGR images and Axial FLAIR scans, in-phase vs. out-of-phase, etc.). Again, the manual for the course was very easy to follow and made the laboratory portion of the course quite enjoyable.
Thursday I was able to treat the slides with hematoxylin and eosin (H&E) stain then leave them overnight to dry, and Friday I was able to look at the slides and plan out the next stages of the project with my research mentor in better detail.
Note: The above image is from http://drsvenkatesan.files.wordpress.com/2009/04/gunther-tulip-ivc-filter.jpg
Saturday, August 14, 2010
Tuesday, August 10, 2010
The experience was not as fun as the rest of summer immersion; apparently watching surgery, imaging, and lab tests are more fun than having them performed on you. Over the course of my treatment I ended up receiving a CT Scan, an MRI, and several x-rays. While all of these imaging procedures were painless and easy the entire process of getting all the appointments did take weeks to complete. I’m not sure where the engineering solution lies, but it seems like this whole process could be streamlined and made more efficient by somehow providing patients quicker access to these tools.
After eventually obtaining all of the imaging needed to decide on a proper course of action two surgeons both suggested the same procedure. They told me my wrist was most likely to heal quickly and correctly if an autograft from my hip was taken and inserted into the space between the fractured bones. This whole mess would then be screwed closed and I’d be left with some cool new hardware and a sweet scar. After going through the information I consented to the procedure and scheduled to have it done.
The process of actually going for the surgery wasn’t too bad. The only difficult part was actually walking into the OR and seeing everybody surrounding me. Having been on the opposite side I never realized how overwhelming the number of people present for a surgery could be. With everyone busy around me doing all sorts of different things; including sticking me with needles and getting scalpels ready it was difficult to remain calm. Eventually they put me under anesthesia though and I’m a little fuzzy on the details from there on.
Recovery has progressed quickly considering all that was done. My wrist is already painless, though the bone will take months in a cast to heal fully. My hip hurt a lot at first, but is also rapidly returning to normal. After surgery I was able to return to work the next day; albeit in a little more pain; and accomplish some paperwork on my research project. While the project is not completely finished I’ve made some considerable progress towards completing it and both advisors in New York and Ithaca seem happy with my progress.
Finally receiving the surgery I needed made an excellent end to summer immersion showing me medicine from the other side. As a patient myself it was immediately much easier to relate. My research project with Dr. Cesarman provided me access to a more clinical side of research then I’ve ever seen and taught me a toolbox of new techniques. Overall my progress has been successful and hopefully with a little more work will lead to publication. Further, we’ve received a small pilot grant through the Weill Cornell Clinical and Translational Pilot center to continue the project.
This program has provided an excellent opportunity and served as a great crash course in medicine. During these seven weeks I was able to learn much more than if I had remained in Ithaca and the experience has helped me develop as a researcher. Thanks to Dr. Wang, Dr. Frayer, Belinda Floyd, and Mitch Cooper for all their time setting this program up; it ran great!
The first task didn’t appear too difficult, but as I began I realized it was going to be hard to detect whether or not my oligonucleotides attached to the gold nanoparticles. I had thought the added mass of the oligonucleotides would cause a shift in the SPR of the particles, but realized because my oligonucleotides were short this wasn’t going to be the case. The aggregation of gold particles would still work; I just needed another method of determining whether the first step of my preparation had completed.
Ultimately I came up with another test to determine whether the oligonucleotides attached to the gold nanoparticles. Specifically, gold nanoparticles react with salt and precipitate out of solution. However, if the surface of the particles is protected by oligonucleotides the particles remain stable in suspension. This stability is also particularly important because DNA hybridization is normally performed in the presence of salt, and will be required for the next step. By running an array of samples changing the concentration of salt and whether or not oligonucleotides had been bound to the gold nanoparticles I was able to confirm their presence. Looking at the figure the gold nanoparticles with no nucleotides rapidly precipitate out of suspension and the solution loses its color. However, once a monolayer of oligonucleotides is attached to the particles they remain in suspension for days.
The next step is to actually test these nanoparticles with DNA from cell lines infected with the virus and see if I can get a color change reaction. So far I’ve been unsuccessful, but I have another week to try. I think the problem might be the proximity of the gold nanoparticles; even though the lengths of DNA I chose closely match the literature I’m worried the particles are still too far. I have some particles with larger radii which I plan on trying which might help alleviate this problem.
This week I also visited the Emergency Room which so far has been one of my favorite places in the hospital. I really enjoyed the fast pace of and was able to see how quick, easy to use technology would really benefit the patients here. I continued in pathology too and observed more patients and cases. I also saw a mortality conference on one case I had followed and was really interested to see all of the information presented together. Another great week in New York!
By week five enough of my materials had arrived to start experiments. I had obtained my gold nanoparticles and my PCR Primers, though not some specific thiol modified oligonucleotides. Just a recap, I’m working on a detection mechanism for Kaposi’s Sarcoma causing herpes virus based on changes in the optical properties of gold nano particles when they aggregate and are packed tightly together. By functionalizing the gold nanoparticles with two different oligonucleotides which are non complimentary and then introducing a third oligonucleotide which is complimentary to both the particles can be caused to aggregate. If this third oligonucleotide is from a virus such a scheme could be used to determine whether or not the virus is present.
Because the thiol modified oligonucleotides hadn’t arrived yet I started with regular oligonucleotides I had ordered with the same sequences which could be used for PCR. Using these PCR Primers I should be able to detect viral DNA using more conventional means and test whether or not my DNA sequences will actually detect KSHV and nothing else. DNA was extracted from two cell lines, one positive for KSHV and one not, and also from a mouse tail clipping. The DNA was prepared for PCR with my primers and the results were run through a gel.
The results came out positive; only the cell line positive for KSHV showed the PCR product while all other cell lines and a water control were negative. The product was the right size given the primers and was seen in both KSHV positive samples.
My clinical experiences continued on as normal this week and I observed more brain dissections and histology. Overall this was another successful week and I was happy to begin making real progress in my research.
Monday, August 9, 2010
I wrapped up my time with Dr. Doty by cataloguing some histology by cut, stain, and spinal structure. I became very familiar with how a vertebrae looks under various types of staining as opposed to how a soft tissue, such as the intervertebral disc would look. It was interesting to learn what types of you staining you might want to use when evaluating different tissues in the body. We were unable to schedule another MRI class, but the workbooks we recieved in addition to the previous training more than made up for it.
I ended the week much the same way as I started the Immersion term: watching surgery and seeing patients. I observed a L5-S1 spinal fusion with the use of a special technology developed by SpineWave. Dr. Hartl used the StaXX XD expandable device to aid in restoring disc space height after the discectomy. This system allows for controlled distraction of the disc space in 1 mm increments via a simple ratcheting device. Additionally, this system is minimally invasive. After the device was implanted, rods and screws were then used to correct scoliosis in the spine.
In just this short immersion term, I have learn much about the in-depth anatomy of the spine, breadth of spinal traumas that extend past the scope of my research project, and have truly gained insight into how my research at Cornell can translate to a beneficial clinical result. I can honestly say that this experience has helped to reshape my thinking about my Ph.D thesis, and I hope for continued collaboration with Dr. Hartl throughout my tenure at Cornell.
The "hook-up" was only mildly uncomfortable: the scalp has to be scoured with alcohol to ensure firm electrode connection. Despite the multitude of connections and wires, it wasn't too cumbersome. However, I experienced a good deal of difficulty falling asleep. This phenomenon is so frequently encountered that it has its own name: the "first night effect." Participants in sleep studies often experience difficulty falling asleep. If the study is conducted for more than a single night, the subjects often acclimate to their new surroundings and sleep soundly. I recall repeatedly being on the verge of sleep only to awaken abruptly with a spastic twitch, much like when trying to stay awake when tired in a lecture. It was as a result of one of these spastic, involuntary twitches that I jerked the finger pulse oximeter loose, prompting one of the technicians to enter to reconnect it. After a fitful night's sleep, I was awakened by one of the technicians. Feeling not at all refreshed, I dressed, walked back to my dormitory, and sunk into a deep sleep.
Later in the day, I was able to sit through the scoring of my sleep quality with one of the technicians. It was definitely a unique ̶ and somewhat unnerving ̶ experience viewing myself on the video. Initially, I had been concerned that I had "failed" the sleep test. Upon waking in the morning, I was firmly convinced that I hadn't slept at all, or at most, only dozed off. I had, in fact, slept for 3 hours and 45 minutes, enough for an assessment to be made.
Much to my surprise and relief, it turns out that I don't have sleep apnea. The index for number of breathing events was well within the normal range. Specifically, my Apnea/Hypopnea (A/H) index was 1.1. Sleep apnea is considered mild if 5-15 such events occur per hour. Moderate in the range of 15-30 events per hour. Severe is above 30 events per hour. While it seems that I am clear of sleep apnea, I was cautioned that sleep apnea tends to be more severe during REM sleep and due to the disturbed nature of my sleep, I experienced only a modest about of REM sleep (about 15 min). However, during this period of REM sleep, no episodes manifested. Also, I stayed on my back (the supine position) for most of the night. As sleep apnea is most severe in the supine position, I don't have to fear the assessment was skewed by my body position.
There was one unnerving breathing episode in which I appeared to stop breathing for an entire minute. The distinction between obstructive and central sleep apnea is an easy one to make. In central apnea, the chest fails to expand (a nervous system condition). In obstructive apnea, the chest continues to rise and fall, but air is unable to be drawn into the lungs. In this one minute cessation of breathing, my chest was seen to rise and fall, so this was an example ̶ and a disconcertingly long one at that ̶ of obstructive sleep apnea. When breathing resumed, I was seen to stir, being momentarily awakened. While it may sound alarming, my saturated blood oxygen level (SaO2) barely dipped in the aftermath of this breathing cessation. The technician explained that in some people, such as those with chronic obstructive pulmonary disease (COPD), such a breathing episode would be followed by a precipitous drop in SaO2. Throughout the course of the night, my SaO2 levels remained in the high 90s. Also, much to my surprise, it turns out that I do not snore.
While sleep apnea was ruled out, I was still left wondering what may account for my daytime drowsiness. Another technician looked at my sleep study and identified it as a textbook case of "delayed sleep phase syndrome." DSPS is a "circadian rhythm sleep disorder, a chronic disorder of the timing of sleep, peak period of alertness, the core body temperature rhythm, hormonal and other daily rhythms relative to societal requirements." People with this condition report being unable to fall asleep until early morning and find it difficult to wake early for their obligations and commitments. There are a number of ways to remedy this condition, mostly through lifestyle modifications. These changes include abstaining from caffeine after noon, not engaging in strenuous physical exercise in the evening, and adherence to a strict sleep regimen. My lifestyle was definitely not following these simple rules as I would routinely consume caffeine throughout the day, lift weights late at night (thinking it might serve to tire me out), and had little regularity in my sleep cycle due to the changes in my workload.
I enjoyed my summer immersion experience at Weill Cornell and have a newfound respect for physicians and support staff who must interact with the sick on a routine basis. I now appreciate just how hectic their lives are and the pressing need that exists for engineers to provide superior devices to shorten wait times when every second matters and to lengthen patients' lives when every extra day is cherished.
Tuesday, August 3, 2010
For my final week here in NYP I worked mostly on my research project since the sodium oxalate I ordered finally came in. So I spent my time in lab testing out different responses to different concentrations of sodium oxalate and calcium chloride. In all my tests whether or not I submerged a stainless steel paperclip in the solution, there was precipitation at some point of calcium oxalate. For most of my tests involving lithogenesis with paper clips I tried to establish a concentrations of .5 mM of sodium oxalate and calcium chloride under constant stirring. My experiments ranged from 2 hours to 24 hours just to see the effects and I am happy to report that the paperclips have all calcified to the degree positively correlated to the experiment run time. Knowing that lithogenic metal undergoes lithogenesis in this experiment helps me establish a positive control for future experiments. I also hope to obtain a calcium ion-selective electrode in the future to help me quantify the calcium ion concentration in solution over time for a more quantitative result as well as take SEM pictures of the crystals formed on the clips. Once this has been established I will move on to testing lithogenesis on various elastomeric biomaterials for potential use in an artificial bladder.
I also saw the last part of a complex surgery this week on a male patient in his 60's who was suffering from renal cancer designated T3c. The tumor had spread all the way from his left kidney up into the inferior vena cava; this was a joint surgery between my mentor Dr. Scherr and Dr. Giradi who was a cardiac surgeon. Briefly, in order to fully remove the tumor they had to divert blood away from the heart using a machine in to stop the heart and allow the doctors to push the tumor out of heart so that they could remove the whole tumor at once. The head was iced and the blood was cooled by the machine to prevent neural damage during this part of the procedure. Once the tumor was removed the doctors worked to reperfuse the heart and warm up the body to allow the patient to natural circulate his blood. They inserted a temporary pacemaker into the patient to help coordinate his pace with his reperfusion.
I would like to sincerely thank my mentor Dr. Scherr and all of his residents for being patient with me and showing a great time in the OR and hospital. I have learned a lot here and of the importance of developing technologies to aid in medical care and treatment, I will carry this motivation forwards with me as I continue on my PhD research career. I would also like to thank Dr. Frayer and Dr. Wang for organizing this whole summer endeavor and treating the whole BME class to a delicious farewell dinner. May all the Cornell BME students prosper in the years to come!!!
Monday, August 2, 2010
In addition, I had a few conference calls with my Mentor and GE to discuss future collaboration this fall, and it looks like I will be getting a paper out of the work I did this summer, and future work I will do this fall.
All in all, this summer has been a great experience. I was able to see Cardiac MRI, Cardiac Electro-physiology, Cardiac Echo (and TEE), open-heart-surgery, rounds in the CCICU, rounds in Cardiac Telemetry, Nuclear Imaging and Nuclear stress tests, and finally a pacemaker implant. Being able to see all of these activities in Cardiology, has definitely helped me gain insight into both how the heart works when healthy and also how the heart works and is fixed in diseased states. I now am more aware of the clinical aspect of my PhD research in Cardiac MRI. Thanks again to Drs. Wang and Frayer for organizing immersion.
Sunday, August 1, 2010
Over the last few weeks I have been able to see how doctors use technology to improve healthcare for their patients. Biomedical engineers do research in technology that has direct applications in the medical field and this experience is not only important for gaining an appreciation and sense of where future developments will be needed, but it also helps to foster collaborations between physicians and students like me. My summer project for example was very applicable to what I will be continuing to study in future research; where I am facing many similar mathematical problems and using many of the same tools. I feel very fortunate that over the summer I was able to observe how technology works with medicine in modern healthcare. I was able to see many aspects of healthcare from seeing patients with Dr. Gauthier, reading neurological images with Dr. Comunale to observing how this kind of information was used in the OR with Dr. Greenfield and Dr. Kaplitt in neurosurgery. Beyond neurology I also saw the importance of imaging modalities in other departments of the hospital; primarily in pathology, urology, the intensive care unit and cardiothoracic surgery. These experiences helped me to understand how doctors in other departments use technology including imaging in their clinical cases. To observe these complicated procedures and clinical activities was no easy task; it required some study on the problems that these doctors face and the vocabulary that these doctors use to communicate their activities and situations. This was probably the most difficult task over the summer. It took some effort to study and ask enough questions about the situations in order to understand enough about the clinical situation to be able to understand the doctors’ point of view and the challenges that they face during their procedures. Once I could understand these challenges I could really see the synergy between the science of medicine and the aid that current technology provides.
My project in a way helps with this goal. My project will help Dr. Gauthier in the research that her group is doing to understand what happens in MS lesions during the progression of the disease; and I may be a contributing author on a paper the group is currently writing on T2 relaxometry. Earlier in the week I was able to present what I was able to accomplish over the summer in a meeting to all of the immersion students and advisors for the program. I was able to show how the fitting algorithms that I was working with work with some of the data that I have in both the mouse scans that were taken earlier in the summer and with some human data.
I am very grateful for the unique opportunity that I was able to have over the summer I have learned so much about a field I otherwise would never have been able to observe. This experience has taught me so much and I am grateful for the generosity and patience that the nurses, surgeons and doctors showed me, Thank you.
My research project from this summer will hopefully lead to a manuscript with both biomedical engineering and clinical aspects: prostate cancer cell line characterizations (MDA-PCa-2b) and circulating tumor cell capture and culture from patient samples with known stages of metastasis, respectively. Dr. Nanus’s lab and our lab have also established collaboration based on this project and I look forward to presenting our results at the upcoming PS-OC conferences.
Again, special thanks go to people who organized the summer immersion and made all these wonderful experience happen: Dr. Yi Wang, Dr. Frayer, Dr. Nanus, Mitch Cooper, and all the doctors and nurses who let me observe in their ORs/ER/NICU.
Although this is the final week of our Summer Immersion, I can confidently say that the insights, skills, and lessons learned from this experience will continue to enrich my career and perspective for many years to come. Not only was I able to observe a variety of surgeries in the OR across a range of departments (plastic, pediatric, cardiac, etc.), but I was also able to witness grand rounds debates, accompany the residents during patient rounds, attend general surgery lectures, visit the ER and NICU, learn to perform MRI scans and other diagnostic tests, and watch patients progress over the 7 week period from a highly invasive surgery to an almost completely healed state. My mentor, Dr. Grant, was an amazing teacher - I could not have asked for a more knowledgeable and enthusiastic guide to help me along my first steps into the clinical world and to help me understand the trials and tribulations of patient care. Moreover, my experience was slightly different than that of my peers because I had the opportunity to visit both Cornell and Columbia medical hospitals and interact with surgeons and residents in both locations. What few of us may realize is that different hospitals do things differently with regards to patient care. Yes, there are institutionalized rules and standards that must be followed…but if you pay attention, you will see that there are numerous subtle differences in the way hospitals design their residency program, the way ER nurses screen walk-in patients in the ER, and even in the way residents and surgeons go about performing surgeries in the OR (in terms of what instruments they prefer to use, how they position the patient, how they plan their incisions, etc.).
Lastly, I would like to thank everyone in the BME department, Cornell Medical Hospital, Columbia Medical Hospital, all my friends, and my clinician mentor Dr. Robert Grant, for supporting my adventure into the clinical and applied side of Biomedical Engineering. Overall, this Summer Immersion was a wonderful, eye-opening experience!
After the dehydration process was completed the each femurs was cut with IsoMet ® 1000 Precision Saw. Using the IsoMet®, each femur was cut at the three different positions: at the middle, at positions 7.0 mm, and 15 mm. Both hands were used for the first cut; later cuts were made by programing the machine. The metal implant was cut at position 7mm and 15mm. The optimal speed was 150 rpm.After cutting each individual femur, they were characterized by using the Scanning Electron Microscope (SEM), histology studies and mechanical testing.
In order to calculate the displacement of the vertebra, a human lumbar spine of a three years old male cadaver was used. Saline solution was used to keep it hydrated. Clay was placed at each end of the vertebra and a metal platform was screwed. A total of 10 rod pins with reflector tape at the end of each rod. The vertebra was placed in the Bionix ®EM Test System.
The clinical summer immersion program at Weill Medical College of Cornell University was a transforming experience. It was able to give me a clear understanding of how people’s lives are impacted through my PhD research. Each surgery and rounds that I attended made a change in my personal career. At the end of the seven weeks, I was able to understand the vocabulary that medical doctors used. Also, I was highly impressed with the advance technology that the HSS and Weill Cornell Medical College are using to improve people’s health.