Standardizing Histocompatibility Testing Protocols
Dr. Robert Liwski is the Medical Director of the HLA Laboratory at the Queen Elizabeth II Health Sciences Centre and Professor of Pathology at Dalhousie University in Halifax, Canada. He completed his PhD in Transplantation Immunology in 1999, his MD degree in 2003 and then went on to complete a fellowship training in Hematological Pathology in 2006, all at Dalhousie University. In 2012 he also completed the American Society for Histocompatibility and Immunogenetics (ASHI) Director Training Fellowship. Currently, Dr. Liwski is a clinician scientist with interests in transplantation immunology, ischemia reperfusion injury and optimization of diagnostic testing in transplantation. In his pursuit of optimizing and standardizing transplantation assays, Dr. Liwski has developed several HLA antibody testing methods, including the Halifaster flow cytometry crossmatch (FCXM) protocol and the Rapid Optimized Single Antigen Bead (ROB) assay.
The Scientist: Dr. Robert Liwski
1. What got you interested in the transplantation immunology field and ultimately motivated you to do a PhD in this subject?
For my undergraduate degree, I completed a major in microbiology & immunology. However, my ultimate goal was to go to medical school, do medical research and become a clinician scientist. With this goal in mind, I knew that after my Bachelor of Science Degree I wanted to complete a PhD before going to medical school. During my undergraduate degree, I took several immunology courses, and the subject on transplantation immunology sparked my interest. I was particularly amazed to learn how a single organ donor could save the lives of several people suffering from organ failure and organ damage. Similarly, in respect to bone marrow transplants, I was fascinated by the idea of being able to take stem cells from a donor and repopulate the entire blood and immune system of the recipient and in the process, potentially curing leukemia or lymphoma. I also liked the fact that this particular area of immunology research has a clear and direct medical and clinical application with the potential for saving many lives. That interest and passion led me to complete a PhD in transplantation immunology.
2. After you completed your PhD, what led you to your current role?
Towards the end of medical school, one starts to think about what specialty to go into. I wanted to be able to use my knowledge and background in transplantation immunology and looked for a medical specialty that would allow this. At the end a specialty that jumped out at me was hematopathology. This is a subspecialty of pathology that allows you to work with diseases of hematopoietic cells, and with patients with a variety of hematological disorders including leukemia, lymphoma and other non-malignant disorders such as anemia. Hematopathology also encompasses transfusion medicine, stem cell transplantation, aspects of immunology such as flow cytometry, and requires an understanding of normal and abnormal immune cells, antibody-antigen interactions and the development of antibodies against allo-antigens. Based on all this, I decided to specialize in hematopathology and completed my residency in Hematological Pathology at Dalhousie University. Becoming the Medical Director of the HLA Laboratory at the Queen Elizabeth II Health Sciences Centre in Halifax seemed a natural extension. I had all this experience in transplantation immunology and hematopathology, and by adding extra training on the assessment of patient-donor histocompatibility, I was able to combine all my areas of expertise in this new role as the Medical Director of an HLA Laboratory.
3. What do you enjoy the most about your current job?
I really enjoy working with a variety of individuals in the laboratory and in the clinic. In the lab, I really enjoy working with technologists. On the clinical side, I work with transplant coordinators and transplant clinicians providing consultation regarding assessment of patient-donor histocompatibility. I also like working as part of a team; in this day and age, you need a variety of backgrounds and expertise in order to successfully carry out scientific and clinical research.
4. What aspect of teaching, mentoring and contributing to histocompatibility community do you find the most fulfilling and why?
In my current role, I mentor technologists and educate other directors and technologists about techniques in histocompatibility. I also share our optimized protocols, explain how we develop these and ultimately help implement these protocols and testing in other laboratories. At the end of the day, education around histocompatibility and providing optimized protocols for histocompatibility testing will improve and impact patient care, which I find extremely rewarding.
5. Your laboratory is located in Halifax, Nova Scotia and you have lived in Halifax since 1989. What do you like most about living in Halifax and why?
Halifax is a capital city of the province of Nova Scotia, yet it is a relatively small city with a really nice community. People here are extremely friendly, which translates into a very collegial atmosphere at the hospital and at the university. Within the laboratory, the different technologists and myself work extremely well together. There is also a very collegial environment within the division of hematopathology between myself, other hematopathologists, the management team, and also the clinical colleagues on the solid organ transplant and bone marrow transplant teams. And of course, Nova Scotia is beautiful, and there are many outdoor activities that you can do here that keep you very busy.
The Science: Histocompatibility
1. Tell us about your research and some of the important findings your team has made.
The focus in our lab is to try to improve and optimize pre-transplant testing by improving histocompatibility testing and, in particular, HLA antibody assays. We have been successful in optimizing the flow cytometry crossmatch (FCXM) assay and developed two protocols: the Halifax and Halifaster protocols. These protocols save time, decrease costs and improve the specificity and reproducibility of the assay. We also applied similar optimization strategies to improve the single-antigen bead HLA-antibody detection assay and were able to significantly reduce the time that it takes to perform this assay while improving reproducibility.
2. Why do you think those findings are significant for the field?
As we start sharing organs nationally and even internationally, it is very important where the testing is performed. If the testing is done in my laboratory or elsewhere, it is important that the results are consistent, so that equitable organ allocation can be improved. In the histocompatibility field the time it takes to perform the testing is also crucial. Testing is time sensitive and improving the turnaround time to provide faster results can directly impact the clinical team to more efficiently select the patients that will undergo transplantation and organize the logistics around the transplantation. A faster turnaround time in testing also shortens ischemia and reperfusion times, which then minimizes adverse outcomes post-transplant.
3. What do you think is the most important advancement in the histocompatibility field in the past 5 years?
A recent advancement and thought that has been going on within the histocompatibility testing field is the idea that HLA molecules should be thought of as a collection of immunogenic epitopes — epitopes that are responsible in part for patient sensitization and then ultimately for graft rejection. Using these epitopes to predict the risk of post-transplant rejection and anti-HLA antibody formation has been a huge advancement. Previously, we were trying to match as many antigens as possible without realizing that some antigen mismatches are much better tolerated than others. We are starting to understand and learn that the antibodies that are formed really depend on particular epitopes found on the surface of HLA molecules. This is a concept that previously was not really being taken under consideration.
4. What are some of the main technical challenges facing the histocompatibility field?
A main challenge that histocompatibility field is facing is the standardization of testing. If you look across North America and Europe, the practice of histocompatibility testing is very different from laboratory to laboratory. Protocols may be different, the cut off values for calling positive vs negative reactions differ and what one laboratory will interpret as clinically significant antibodies may also be very different. It is even possible for different laboratories to get conflicting results, depending on what assay and what approach they use. We, the histocompatibility community, need to standardize assays across all laboratories and come to a consensus as to which antibodies are clinically significant and which ones are not. Perhaps a way to address this challenge is to use these standardized assays in multicenter research trials and get results that will give us a better idea of which antibodies are truly clinically significant and which may not be, and which characteristics make an antibody clinically significant.
Lymphocyte Isolation with EasySep™ Direct
1. In your most recently developed and optimized rapid flow cytometry crossmatch (FCXM) protocol, the Halifaster FCXM protocol, the cell isolation step is performed using EasySep™ Direct lymphocyte enrichment technology. Why did you decide to incorporate the EasySep™ Direct Human Total Lymphocyte Isolation Kit into your protocol?
As you know, majority of labs still use lymphocyte separation medium (LSM)-based methods that use a density medium such as Ficoll™ or Lympholyte®. Using this method to isolate cells from blood samples is very time consuming, tricky to perform and the final yield of cells and lymphocyte purity is variable. The lymphocyte purity also depends on the donor sample, and we have found that lymphocyte purity ranges from 15% to 80%. The research we have done in the laboratory showed that this variability of purity can affect your crossmatch results. Neutrophils and monocytes express high levels of HLA, which will cause HLA antibodies to bind to these cells instead of binding to the lymphocytes. That means if the cell population used for the crossmatch assay does not consist of pure lymphocytes and there are contaminating neutrophils and/or monocytes, the crossmatch results will be either weaker than what they actually are or even negative. With this in mind, we felt it would be really important to have a way of isolating pure lymphocytes, in a timely fashion and in a manner that removes other contaminating cells without affecting lymphocytes in any way. EasySep™ Direct is a perfect solution for that: it is rapid, it removes all other cells including red blood cells (RBCs) and you avoid having to perform any lysis step, which could negatively affect the lymphocytes. The isolation method used by EasySep™ Direct is negative selection, which means that lymphocytes are never altered in any way by the reagent.
2. Before using EasySep Direct, what methods of isolation were you using?
We were using lymphocyte separation medium (LSM)-based method with Lympholyte® at first. To improve lymphocyte purity we used the RosetteSep™ and Sepmate™ system. We then moved away from density gradient centrifugation techniques and are now using the immunomagnetic system EasySep™ Direct, which is very easy to perform and has no technical nuances. EasySep™ Direct is very effective at removing other blood cells and yields consistent purity that is at 90-95%.
3. What aspect of using EasySep™ Direct technology is appealing to you and how has that enabled your research?
As mentioned before, the fact that EasySep™ Direct is rapid, removes RBCs without a lysis step and yields high lymphocyte purity is very appealing. It is also easier to train lab members how to use EasySep™ Direct for cell isolation compared to LSM. When using EasySep™ Direct, there is not much that can go wrong, the protocol is very easy to follow and it is difficult to negatively impact your isolation.
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