In the search for a fast and convenient way to diagnose autoimmune damage to the small nerve fibres, scientists at St Petersburg University have attempted to look at the cornea by using confocal microscopy.

2019 11 25 natalya basanceva 1

Natalia Gavrilova, Assistant Lecturer in the Department of Faculty Therapy at St Petersburg University and neurologist at St Petersburg Research Institute of Phthisiopulmonology

Natalia Gavrilova and her colleagues at the Laboratory of the Mosaic of Autoimmunity at St Petersburg University are continuing to study the ways to diagnose small fibre neuropathy. She is Assistant Lecturer in the Department of Faculty Therapy at St Petersburg University and neurologist at St Petersburg Research Institute of Phthisiopulmonology. Small fibre neuropathy occurs when the small nerve fibres in all our organs and tissues are damaged in cases of immune system overactivity. This condition can cause various neurological symptoms.

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' A healthy volunteer': parallel straight nerve trunks of nerve fibres (thick lines) with few branches (thin lines), few Langerhans cells (white shapeless dots).

In 2019, scientists had already been studying what the skin biopsy techniques could offer for the diagnosis of small fibre neuropathology. Skin biopsy is the most common diagnostic test for this damage. ‘Although skin biopsy is considered as a “gold standard” in diagnosing autoimmune pathologies of the small nerve fibres, it nevertheless has a number of disadvantages,’ said Natalia Gavrilova. ‘First, skin biopsy is a medical procedure to remove a sample of the skin. Consequently, some complications may occur. Second, no follow-up examination is possible as new skin will grow on the site where the sample was removed. Next time you will have to take a sample from another area where the nerve fibre pattern might be completely different. Third, skin biopsy must be performed by an experienced specialist who is able to perform all necessary tests. Finally, the skin biopsy techniques have not yet been standardised. In other words, there is no standard procedure that would ensure reliability and accuracy of the results. There is evidence that a sample from the ankle skin can be used for biopsy. Yet damage to small nerve fibres has a disseminated nature. If neuropathy affects a face, biopsy from the ankle would be of no use. The conclusion to be drawn is obvious. We need to find a new diagnostic technique.’

New technique

What the team of scientists at St Petersburg University has been studying is corneal confocal microscopy. They intend to gain a better understanding of whether this technique can be used as an additional or main tool to diagnose autoimmune damage to the small nerve fibres.

Corneal confocal microscopy is a non-invasive technique to detect even subtle changes in the small fibres. It is painless and can be performed several times to observe and check on the progress of treatment. Additionally, this technique permits visualisation of the entire corneal thickness in both eyes. This cannot be done in other cases. For example, the shin has a wide skin surface. You can fail to detect damage in one biopsy site, yet obtain positive test results after examining another biopsy sample taken from the shin. The only drawback of the corneal confocal microscopy is that it cannot be used for quantitative assessment of the cytokine concentrations and factors of tissue inflammation which can trigger autoimmune diseases. The confocal microscopy images provide visualisation of the Langerhans cells (i.e. cells that reside in the epidermis as a dense network of immune system sentinels. Notes by an editor). Depending on the results of the quantitative assessment of these cells, we can detect inflammatory processes and perform additional tests.

Read more about skin biopsy for diagnosing sarcoidosis in ‘Our immune system is both beautiful and dangerous’ in St Petersburg University, Issue 05–06, 2019).

‘Confocal microscopy is based on the principle of juxtaposition of two lenses. The first lens is placed on the cornea of the patient, while a doctor looks through the second lens.

Juxtaposition of optical beams from each lens produces a 3D-image in real time,’ said Mariia Lukashenko, a sixth-year student in general medicine at St Petersburg University and a member of the team. ‘When using confocal microscopy, you can apply various modules that enable a better visualisation of the object. For example, in our research we use a special retina module. It permits the study of all five corneal layers.’

How does it work? First, a scientist should get an image of the cornea. Then, you should perform quantitative assessment across four main indicators: length of the nerve fibres; breadth of the fibres; the number of branches from the trunk and tortuosity; and imaging depth. Based on the obtained results, the scientist can assess the extent of damage to small nerve fibres. Initially, the assessment procedure was performed by enlarging an image by zooming in Adobe Photoshop, as Mariia Lukashenko explained. The whole procedure could take three hours. Later, scientists contacted Professor Rayaz Malik at the Weill Cornell Medicine in Qatar. He had been studying confocal microscopy for a long time. He helped our scientists know how to use a special software solution to count small fibres. Now the whole procedure takes much less time.

‘This software solution assigns a coefficient to each of the main indicators. All you have to do is to mark necessary areas and the programme will produce numerical results. The programme is nevertheless only for those who have knowledge and expertise how to work with it,’ said Natalia Gavrilova. ‘The team headed by Professor Rayaz Malik is currently developing a new algorithm that is based on artificial intelligence. It is intended to make the whole procedure faster and easier. For example, you will not have to mark areas on the image.’

Difficult diagnosis

The scientists are working with patients who suffer from fibromyalgia or chronic fatigue syndrome. Fibromyalgia is a disorder characterised by widespread musculoskeletal pain. Chronic fatigue syndrome is a disorder characterised by extreme fatigue or tiredness accompanied by cardio-vascular disorders. Seemingly, these are two completely different pathologies. Yet they have much in common, as scientists revealed at the initial stages of their research. The small nerve fibres surround blood vessels and regulate blood flow in tissues. They also carry pain signals. Any damage to the small nerve fibres in cases of immune system overactivity is associated with pain or orthostatic symptoms (e.g. dizziness) that are usually associated with chronic fatigue syndrome.

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'A patient with chronic fatigue syndrome': only two trunks that are tortuous and have many different branches, there are many Langerhans cells.

‘Both fibromyalgia and chronic fatigue syndrome are not studied at all. Doctors tend to avoid diagnosing these conditions in patients. Patients consequently visit various doctors. This impedes proper treatment. People who suffer from chronic fatigue syndrome are especially at risk. As a rule, they have lots of tests, yet all results are normal. You cannot diagnose this syndrome by appearance. Patients don’t have any symptoms of pain. All they suffer from is dizziness and differences in pulse rates according to the position of their body. For example, when they stand up, their pulse rate can increase up to 120 beats per minute,’ said Natalia Gavrilova. ‘We are planning to define precisely clinical criteria for diagnosing both conditions.’ It is vitally important, as she explained. People who suffer from fibromyalgia will be able to be registered as a disabled person. They experience acute pain that sometimes cannot be relieved by medication. Working is extremely difficult for them. If we define morphological criteria more precisely to diagnose these conditions, we can include these disorders in the list of conditions to cover patients as disabled.

What next?

The scientists are planning to study patients by using a wide range of various techniques. They have already surveyed volunteers by using special questionnaires; they get volunteers ready for skin biopsy and blood tests to check their immune system. Additionally, they are going to take other tests. Among them is an audiometry exam to test both the intensity and the tone of sounds. The tests are also associated with how well the small nerve fibres work.

The research by scientists at St Petersburg University is intended to extent what we know about corneal confocal microscopy. They have already managed to make a most comprehensive list of patients. It includes 70 people across Russia. Previous research projects were far from being able to study so many patients.

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'A patient with fibromyalgia': you cannot identify any trunk, you can see only branches; there are an incredible number of Langerhans cells.

‘All in all, what we are driving at is to analyse whether the results of skin biopsy are in agreement with the results of confocal microscopy, and to compare them with the results of the laboratory tests. Additionally, we are going to assess the confocal microscopy technique in terms of whether we can use it as the main tool to diagnose small fibre neuropathy without biopsy,’ said Mariia Lukashenko. ‘If we are even half successful, we will be able to use this tool beyond diagnosing autoimmune diseases.’

Confocal microscopy can reveal eye disorders in patients with diabetes, she explained. Among the complications of diabetes are both diabetic retinopathy and damage to the small nerve fibres and their functioning. These conditions are associated with other clinical symptoms and can be revealed by using the technique of confocal microscopy.