Pathology: detecting lymphedema with 3D microscopy
Pathology: detecting lymphedema with 3D microscopy
Interview with Dr. René Hägerling, "Cells in Motion" Cluster of Excellence, Max Planck Institute for Molecular Biomedicine, Westfälische Wilhelms-Universität Münster (University of Münster)
According to the WHO, 300 million people throughout the world are affected by lymphedema. This condition occurs when fluid that flows between cells is no longer transported back into the blood circulation and accumulates in the skin. Triggers can be surgeries, injuries or genetic defects for example. A new microscopy technique could now also indicate the causes.
Dr. René Hägerling
In this interview with MEDICA-tradefair.com, Dr. René Hägerling talks about the VIPAR imaging technique ("volume information-based histopathological analysis by 3D reconstruction and data extraction") and explains how it can help patients with lymphedema.
Dr. Hägerling, you have developed the VIPAR imaging technique in the task force headed by Professor Friedemann Kiefer. How does it work?
Dr. René Hägerling: This technique allows us to examine tissue samples as a whole. We process them so that we can perform antibody-based immunofluorescence staining (immunostaining). We subsequently clear the sample, rendering it optically transparent.
Using light sheet microscopy, we produce between 3,000 and 5,000 optical sections of the sample measuring approximately one micron in thickness. The computer then creates a 3D reconstruction using these individual optical sections. We subsequently conduct a segmentation analysis using an algorithm, where we extract data on the structures we are particularly interested in – the blood and lymphatic vessels. We automatically obtain vessel parameters such as branching, diameter or the volume of vessels in the entire sample.
In a recently conducted study, we have provided evidence of lumenized lymphatic vessels, that being vessels that are filled with fluid but were not interconnected. This type of condition prevents fluid evacuation from the tissue and consequently causes lymphedema. You were unable to illustrate this using the traditional histological sample preparation and analysis that is currently the gold standard and were thus also unable to detect the underlying pathology.
What progress are you able to achievewith this technique?
Hägerling: For the first time ever, VIPAR allows us to answer questions pertaining to lymphedema patients that we are presently still unable to examine sufficiently. The hospital currently applies two techniques that are unable to conclusively clarify the underlying pathology at the cellular level.
In lymphoscintigraphy, you measure how much contrast agent is transported within two hours from the hands or feet to the lymph nodes in the armpits or groin, respectively. This only lets you make a general statement on whether there is lymphatic fluid transport within the vessels or not.
The other technique refers to the histology and works with the same type and parameters of skin biopsy we also use with VIPAR. However, with this approach, only a few individual sections are analyzed and used to make a determination. This is extremely difficult, particularly since you lack three-dimensional data. The fragmentation of lymphatic vessels we were able to detect in our patient cannot be identified with the standard methods for example. For the first time ever, we now have the chance to truly understand this disease pathology.
In comparison to the classical-histological analysis (A), which provides only two-dimensional information, the spatial arrangement of blood and lymph vessels in a human skin sample can be visualized by means of light sheet microscopy and VIPAR (B).
What ultimate diagnostic conclusion can you draw from the skin biopsies?
Hägerling: Currently there is no curative treatment for lymphedema and especially no imaging technique that is able to adequately depict and identify the pathomechanism at the capillary level. For the first time ever, our 3D microscopy technique allows us to identify and classify patients as well as the causes for their disease in greater detail. It enables us to determine the potential treatment methods for individual patients, as is the case with a current clinical trial that examines the use of immunosuppressive agents for lymphedema. Our method permits us to determine whether a patient is suited for this treatment or not. For example, a patient with fragmented lymphatic vessels would definitely not benefit from the use of an immunosuppressant drug whereas a patient with a different underlying vessel structure actually might.
We can also help optimize treatment. Most likely, patients with highly fragmented vessels would receive more drainage combined with compression therapy to halt the progression of chronic lymphedema. Meanwhile, patients exhibiting mild swelling but whose lymphatic system is similar to a healthy system are assumed to have a more moderate risk of progression given the right treatment.
Having said that, the disease pathology overall has not been conclusively determined yet. That’s also why our approach is initially essential to understand the disease and thus gain new insights for the development of pharmacological interventions. In the long run, I also think that we will be able to use this technique for a number of additional types of tissues and problems.
What are some remaining challenges for you?
Hägerling: On the one hand, we are dealing with a vast amount of data. A biopsy requires between 50 and 100 gigabytes of disk space per picture. Managing the data volume is a challenge, especially as it pertains to a broad application. Having said that, I am confident that there will be a solution sometime soon.
On the other hand, we would obviously like to eventually establish this analysis as an expansion or possibly even as the gold standard in the hospital. However, we still have to conduct further studies before this happens. I am going to conduct a first major study in the coming weeks in one of the world’s leading specialist hospitals in London to test the applicability of our technique and refine it. In this case, we examine a selected cohort of patients with hereditary lymphedema using VIPAR to learn more about the structures of the vessels and their pathomechanism. Since these are highly debated questions for these patients and the corresponding mouse model is possibly not transferable to humans, we are very curious about the new insights VIPAR will provide – especially as it pertains to the development of new treatment approaches.
In the short to medium term, this diagnostic approach should also be accessible to more patients. That is why I could also envision offering some type of diagnostic service for 3D pathology since the technique is presently still too sophisticated and expensive to comprehensively implement it in all pathology institutes. We would like to use VIPAR to help identify new patient cohorts, find out more about the disorder and ultimately support the development of new treatment approaches. After all, without a detailed understanding of pathology, the development of a drug therapy to help the more than 300 million patients around the world is very restricted or rather difficult to do.