Liver transplants

3 a.m. in the morning. A liver is transported under emergency conditions to the Paul Brousse hospital’s Hepatobiliary Centre in Villejuif, for transplantation. But before the organ can be transplanted, it is crucial to determine whether or not the liver is healthy. This is the pathologist’s work: she analyses a dyed sample of the organ under a microscope. The transplantation depends on her decision. But it’s an imperfect approach. After brainstorming ways of making the analysis of transplants more reliable, researchers and clinicians turned to SOLEIL.

A liver under the infrared beam

PAUL BROUSSE HOSPITAL Denis Castaing, Surgeon at Paul Brousse hospital
We do about 130 to 140 transplants every year. 
Since there is a shortage of transplantable organs, we must use so-called “marginal” organs that are not entirely normal on all preoperative examinations. Our dilemma is to determine which of these marginal organs can be used and which must not be used.

In case of doubt, a small sample of the donor liver is sent to the hospital’s testing laboratory located two floors below the operating room. The sample is frozen, cut into slices, dyed and examined by the expert eye of a pathologist.

 
[Telephone conversation between Catherine Guettier and the surgeon] 
Catherine Guettier : The organ appears to be OK; macrovacuolar steatosis is only at 20% and microvesicular steatosis at 30%, with no other noteworthy anomalies, and no fibrosis. I would say it's doable and that the liver can be transplanted. 
Surgeon: Excellent, we can go ahead with the transplant then. Thank you very much.

Catherine Guettier, Anatomopathologist at Paul Brousse hospital
The main issue we must consider is the liver’s degree of steatosis. Steatosis is the accumulation of lipids in the liver. This type of lesion is fairly frequent and may have many causes, including alcohol abuse, diabetes or simply being overweight. Many people suffering from these disorders are likely to have a fatty liver. 

Steatosis leads to the presence of vacuoles, which are round enclosures inside the liver cells. When the liver is excessively steatotic, there is a very real risk that the organ may not resume functioning after the transplant or may begin functioning too slowly, too late and with difficulty, placing the patient in a critical situation.

Despite their experience, it is difficult for pathologists to give reliable figures. They must act very rapidly; ideally, the liver should be transplanted within eight hours of its removal from the donor. And the diagnosis must be as precise as possible, using the human eye as the only analysis tool.

Catherine Guettier 
Our approach is quite imperfect due to the limitations of the human eye and the relatively poor quality of the liver sample that we must assess for steatosis, given the degree of urgency. If we wanted to do a standard evaluation, it would take 24 hours and it's absolutely impossible to wait that long for a liver transplant.

The hospital's research laboratory, which develops new diagnostic tools and new treatments for liver disease, decided to take a closer look at this issue. After brainstorming ways of making the analysis of transplantable organs more reliable, researchers and clinicians came up with the idea or using a state-of-the-art research instrument: the synchrotron.

François Le Naour, Biologist at Inserm 
When SOLEIL synchrotron began operations, I thought it might be interesting to explore the use of light and the quite specific characteristics of this particular form of light to approach tissue composition, particularly liver tissue – since this is what the laboratory and the centre were studying – and also to explore the pathological aspects of the liver, including steatosis and other pathological lesions.

SOLEIL
The hospital's scientists therefore decided to enlist the aid of the SOLEIL synchrotron located on the Saclay plateau. This immense facility, 113 metres in diameter, circulates high-energy electrons at a speed of nearly 300,000 km/s, harnessing the light released during this frenzied process. 
This light, which is 10,000 times more intense than sunlight, is what is known as “synchrotron radiation”. Once produced, this radiation is guided towards a score of outlets and used at different wavelengths by scientists to explore matter on an infinitely small scale. 

On the synchrotron’s SMIS beamline, researchers use infrared wavelengths. This light is used to determine the composition of the samples being analysed (the lipid content of cells, for example).

Paul Dumas, Physicist at SOLEIL synchrotron (SMIS beamline) 
SMIS has the advantage of being able to produce photons in the infrared spectrum in very small sizes and can analyse tiny particles on the order of several microns. 
This is typical for the analysis of liver sections. Since we have a very high resolution beam, we are able to analyse each little component of the liver section individually and see its composition for each analysis.

Liver sections similar to those examined by Catherine Guettier at Paul Brousse hospital are placed under the microscope of the SMIS beamline and analysed with its extremely intense, fine infrared beam.

[Conversation between Paul Dumas and François Le Naour] 
Paul Dumas : Move in on that large vesicle you wanted to see..., there; typically you will only see lipids... 
We can make a chemical image of this composition to see where the lipids are located; we’ll do a mapping around this macrovesicle... 
Ah, it’s a nice one... Your macrovesicle is over there....  
François Le Naour : That’s really very surprising... 
Paul Dumas : There are lipids everywhere, in the vesicle of course, but also all around...

François Le Naour 
Thanks to the synchrotron’s resolution and the infrared spectrum, we were able to explore the inside of the vesicles and we found that it was very rich in lipids and in certain molecular species, which was in some ways to be expected. 
But we were also able to explore around the vesicles and in normal-appearing tissue, which in reality wasn’t normal at all, but full of lipids.

Paul Dumas 
This is an image of the lipids in this liver section. We not only find them in the vesicles, but also elsewhere in the liver. That gave us an indication that there was an abnormal concentration of lipids in the liver that we didn’t see, which the infrared helped us identify inside the liver tissues.

Infrared light provides an extremely precise view of lipids – so precise in fact, that it is conceivable to determine the quantity of lipids present on the basis of a simple image of the liver. The researchers realised that infrared analysis might be the ideal tool to diagnose transplantable organs. After experimenting for several months, their intuition was confirmed. 
They then posed a new question: is it possible to obtain the same results with a small infrared microscope in the laboratory?

François Le Naour 
It is obviously not possible to bring the hospital to the synchrotron or construct a synchrotron in the hospital!

Dozens of liver samples, with known lipid levels, were then analysed with a small infrared microscope. This enabled the researchers to show that the resolution of this small instrument was sufficient to reliably determine the degree of steatosis. And today, this instrument is operational.

[Conversation between Paul Dumas and François Le Naour] 
Paul Dumas : This small portable device is easily adaptable to the laboratory; there is really no need for the sophistication of a synchrotron. It is relatively simple and quick to use, making it a diagnostic tool that is easy for clinicians to set up and manipulate. 
François Le Naour : So it seems the instrument will come to the hospital and not the other way around! 


François Le Naour 
The idea is to reproduce the same conditions as for the procedure currently practiced in the hospital, using the same tissue sections in the same thickness, but instead of dyeing the sample, we simply analyse it under the microscope. It takes the same amount of time – just a few minutes –, but it provides an objective view of the lipid levels measured in transplantable organs. 


PAUL BROUSSE HOSPITAL
Just four years after the research began, the new microscope is ready. It will be transferred to the Paul Brousse hospital in a few weeks.

Catherine Guettier 
We are looking forward to receiving this instrument that will help us make an accurate diagnosis of the level of steatosis. We are well aware of our shortcomings in this regard. This will reduce the pathologist’s stress level and help him or her provide the surgeon with reliable information.

Denis Castaing 
Any diagnostic tool that can help us determine whether or not a marginal liver will function properly is extremely important. 
We expect this system to help us obtain an accurate diagnosis and predict which organs will function properly and which will not, thus providing an invaluable benefit for the patient.