Autoimmunity and automation, a reality or not?

Autoimmunity testing has traditionally been very labour intensive, and hard to automate.  Has technology caught up fast enough to meet the growing demands of this expanding discipline?

Autoimmunity is an ever-expanding field, with an increasing number of pathological conditions identified in which antibodies are directed against normal components of tissues and organs. These antibodies are known as pathological autoantibodies, or autoantibodies for short. They may be directed against individual organs, or groups of related endocrine organs (in which case they are known as organ-specific), or they may have multi-system involvement (non-organ-specific).

Traditionally autoantibodies have been identified by reactions against tissues and cells, using the technique of indirect immunofluorescence (IIF) microscopy. This technique provides a great deal of valuable information regarding which parts of the cell or tissue the autoantibodies are directed against. However it is time consuming, and subjective, and therefore has been extremely difficult to standardise and automate.

Although the process of preparing the IIF slides can be automated, the interpretation of the IIF patterns still requires a skilled laboratory technician. This has lead to many of the tests being transferred into the ELISA (Enzyme-Linked Immunosorbent Assay) format, which requires less interpretation and is far easier to automate. With many assays now in the ELISA format, automation has been a must for many laboratories as the technique, although straightforward, is still very labour intensive and prone to mistakes when done manually.

The need for automation of IIF techniques and ELISA techniques has lead to many automated systems appearing from commercial companies with an interest in this area. Many of these companies have similar interests already in the infectious disease field and have simply adapted existing technology. For example automated ELISA processors, which were already in existence in virology laboratories, have been adapted and developed to run autoimmune assays.

At the same time, companies with an expertise in liquid handling and pipetting systems have developed automation for IIF. Often however, the systems have been quite inflexible - sometimes due to software and sometimes their capacity. This has lead in recent times to a whole new generation of IIF systems appearing on the market.

Now with automation for ELISA and Immunofluorescence widely available from companies, has the problem of automated autoimmunity tests therefore been resolved? 

In many respects it has, and the automation available now is generally robust, and provides an invaluable tool for laboratories with ever increasing workloads and ever decreasing staff and resources. However, until very recently, companies with an interest in this field had still not developed a complete system for the autoimmune laboratory. One that can handle the complete workload, whilst remaining easy to use, and flexible.

Such systems need to be large enough to get through the daily workload of a laboratory, and need to be able to handle both ELISA and IIF assays. They need to be user friendly, and ideally “open”. An open system can accommodate assays from companies other than the manufacturer and supplier of the instrument. Although this is not always popular with the supplier, it does allow the laboratory to have the freedom to choose different assays from different manufacturers.

An example of this type of instrument is the ZENIT SP+, an ELISA and IIF analyser manufactured specifically for the autoimmune market. The system, from A. Menarini Diagnostics, is an automated system that can handle up to 25 IIF slides at any one time, and up to 6 ELISA plates. By allowing both ELISA and IIF to be run simultaneously, the number of tests than can be done on any one patient sample is increased. The system has a large capacity, accommodating 160 samples (both primary and secondary tubes), has bar-coding and the ability to be interfaced.

With 4 fixed probes, the system overcomes the problem of sample transfer to plate time, and can typically manipulate the samples and transfer to a full 96 well plate in under 8 minutes. This speeds up the whole process, and eliminates front to back drift.  All dilutions can be dealt with onboard, either in deep well tubes, or in 4 microtitre trays, for titrations. To cut down on washing time, the system utilises the 8-way ELISA wash head which washes the IIF slides by immersion in specially designed carrier trays, rather than using conventional well by well washing. This technique, which follows closely how slides would be washed manually, speeds up the process and enables multiple slide formats to be used within a run.

The ZENIT SP+ is one of a new breed of instruments that is allowing the autoimmune laboratory to cope with ever increasing demands, and an ever expanding repertoire of tests. With companies investing in instruments like this it is easy to see how autoimmunity - once a niche discipline - has become one of the fastest growing areas of laboratory medicine in the new millennium.

By James Webber, Product Specialist, A. Menarini Diagnostics

Autoimmunity: The body’s friendly fire

The immune system is one of the great wonders of the biological world. Like General Custer and his famous last stand, the system is a past master at battling against the odds. Able to defend an organism from an immensely diverse range of pathogens, it is a system of specialised cells and organs that does not just blindly attack all that it comes into contact with, but rather learns from previous encounters and adapts its response. It is the body’s ‘smart missile’, a strategic arsenal that can pick its targets with accuracy and efficiency.
 
However, like that famous last stand, sometimes the battle does not work out how it was planned.

To function correctly, the immune system must be able to recognise molecules native to the host organism and resist from attacking them. This is known as tolerance, but if it goes wrong the system can become autoimmune and begin to wage war against its own host.

There are around 80 different autoimmune disorders ranging in severity from mild to disabling, depending on which system of the body is under attack and to what degree. There is generally no cure, and for unknown reasons, women are more susceptible than men, particularly during their childbearing years.

So why does the body turn on its self?

Of course, the system does not normally respond to self antigens. This is because when generating its immune response repertoires - identifying its targets - in the lymphoid organs, thymus and bone marrow, all the components that are targeted towards the body’s own antigens are deleted. This deletion, or tolerance, is divided up into central and peripheral, with central tolerance removing most of the self-reactive components by apoptosis.

Particularly implicated in this switch to self-attack are T lymphocytes. Also known as T cells, they are a subset of lymphocytes that play a large role in the immune response. The abbreviation ‘T’ stands for thymus, the organ in which their final stage of development occurs. Indeed, it is in the thymus that T cells targeted to the tissues of the body are destroyed – central tolerance.

In essence, during its development, the immune system it is armed with T cells that target a huge variety of antigens, including those on the surface of the body’s own tissues. It is these T cells that the system needs to delete, and does so by presenting them with self-antigens. If they recognise these antigens, they are killed by apoptosis. About 98% of T cells die in this way in the thymus, with only 2% surviving to maturity.

However, even with this strict dress code, sometimes these self-attacking T cells can sneak past and begin to attack the body. Autoantibodies are then formed as the rogue T cells instruct B lymphocytes (B cells) to make antibodies against particular tissues. There is a back up system – peripheral tolerance – but if this does not stop the rogue T cells then autoimmune disease will develop.

What actually causes the tolerance mechanism to miss certain self-targeting T cells is still not known, but risk factors include genetics, environment and infections.

By Phil Prime, Assistant Editor, Laboratory News