The MELISA® test A revolutionary blood test used to improve diagnosis and

treatment of chronic disease

Metal allergy plays an important role in a variety of medical specialties, such as: • Orthopedics and

implants • Dentistry • Occupational medicine

and environmental health

• Diseases of immunologic origin

• Infectious diseases

“Our case demonstrated the need for proper investigation with tests such as MELISA® and also the need for proper history in all such patients.”

Orthopaedic surgery in a patient with metal sensitivity. Adala R et al. Aesthet Surg. 2011 Jan-Apr; 4(1): 67-68.

A licensing opportunity for Biohellenica The MELISA® test provides important information for patients suffering from chronic degenerative diseases, including chronic fatigue syndrome (CFS), fibromyalgia and multiple sclerosis (MS). It is virtually impossible to avoid exposure to environmental pollutants, such as metals like nickel, mercury and titanium. The most common sources of exposure are dental materials, orthopedic implants and food additives.

With increasing awareness about the possible side-effects from metal exposure in sensitive individuals comes a growing demand for the MELISA® test across the world. In Greece we work with several clinics that send patients for MELISA® testing abroad, but the time and cost for transport limits the demand. All licensed MELISA® laboratories receive access to our contact network and comprehensive marketing support; from ready-made marketing materials to constant exposure through our website www.melisa.org and through lectures and international conferences.

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The effect of metals on health The adverse effect metals may have on human health is a growing area of research and a connection between metals and the onset or worsening of symptoms has been documented by both clinics and in peer reviewed literature. Metal allergy is usually diagnosed by patch testing, but this can be problematic when testing irritative substances as mercury, or substances with large particles like titanium dioxide. Using MELISA® avoids many of obstacles of patch testing.

Chronic exposure to metals in jewellery, dental implants and restorations, cosmetics or joint prostheses can sensitize genetically predisposed individuals and induce a so-called Type IV allergic reaction (cellular type hypersensitivity).

Metals are potential allergens. The binding of metal ions to proteins can alter the protein activity and surface structure of these enzymes, thereby inducing local or systemic symptoms characteristic of allergic reactions or autoimmune diseases.

In contrast to a Type I allergy, which is mediated by IgE antibodies, a Type IV allergic reaction is mediated by T lymphocytes that have had prior contact to a given allergen (memory lymphocytes). Following renewed contact with the sensitizing allergen, these memory lymphocytes respond by enlarging (lymphoblast transformation) and dividing (proliferation). The newly formed cells (effector cells) together with their secreted cytokines mediate the resulting inflammatory reaction.

It is important to differentiate between metal allergy and metal toxicity. The MELISA® test does not measure the levels of metals in a patient’s body; it measures whether the patient is allergic to metals. For example, hair samples may show levels of mercury or other substances which are below the official “safe limit” – but the patient can still be allergic. For allergic individuals, there is no such thing as a “safe” limit. Even a minute amount of a substance may pose danger in an allergic individual.

A well-known allergen, nickel is present in some dental materials and nickel-containing alloys. Such exposure can cause serious health problems in nickel-allergic patients. Nickel is also present in cheap jewelry and many household items. Nickel-allergic patients may benefit from a low nickel diet, mainly avoiding foods such as nuts, beans and oats.

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Orthopedic surgery and implants Metals are found in body implants such as hip joints, knee prosthesis, screws, pacemakers, silicone breast implants, cochlear implants and dental implants. In the majority of patients, these implants are biocompatible. However, in sensitive patients, the implants may induce side-effects such as pain, dermatitis, cutaneous swelling, impaired wound healing, bone infections and implant loosening. Less knows is that even excessive fatigue can be due to chronic inflammation due to metal ions.

Although bacterial infection is often the cause of implant rejection, sometimes no bacteria are detected despite of inflammation. These adverse effects are mediated by so called wearing products of implant materials. Nickel, cobalt and chromium are known metal allergens.

Recently, the cases of titanium allergy have also been described in the literature. Titanium is a transition metal and thus may function as a hapten and trigger cellular hypersensitivity. Since titanium is used as white pigment in toothpaste, cosmetics and medicaments, the sensitization of susceptible individuals is possible.

Dentistry Mucosal changes around dental metal fillings such as oral lichen may be due to an allergic reaction to the metal component. Replacement of fillings in metal-sensitive patients leads to disappearance of the symptoms (Prochazkova et al. 2004).

Allergic reactions to metals used in dental prosthesis, implants (for example titanium implants, see picture above right) and braces can be measured in the MELISA® test. Symptoms may not always be local but can be systemic and include inflammation in different body parts.

Patients with known clinical metal sensitivity such as adverse reactions to gold and nickel containing earrings should consider non-metallic restorative materials.

Occupational medicine and environmental health The MELISA® test can be used to screen workers who are exposed to metals, chemicals or other allergens at work. If sensitivity is detected, the worker should be placed in an allergen-free environment to avoid the development of sometimes serious diseases.

In the case of berylliosis, a sarcoidosis-like disease, a beryllium-specific blood test is used as a golden standard by US industry to screen workers for allergy against beryllium. It has been found that asymptomatic workers with memory cells to beryllium will - if beryllium exposure continues - develop a serious lung disease. This will not happen if asymptomatic beryllium-sensitized workers are relocated to a beryllium-free environment. In addition to lifelong suffering, considerable savings in sick leave costs are obtained. Certain occupations pose a special risk for inducing metal allergy. These include construction workers, miners, electricians, rubber/wood/paper/ textile industry workers, dentists, hairdressers and painters.

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Diseases of immunologic origin Metal allergy is common in patients with various diseases such as skin diseases (psoriasis, eczema), autoimmune diseases (Multiple Sclerosis, thyroiditis, Sjögren’s disease) and gastrointestinal diseases. Patients with symptoms of profound fatigue of unclear etiology (Chronic Fatigue Syndrome, Myalgic Encephalitis) often suffer from metal allergy induced by dental metals. Candida allergy, drug allergy (such as penicillin allergy) and allergy to chemicals (such as formaldehyde) can also be tested. Metal allergy has also been implicated in autistic disorders and allergy against thimerosal (a mercury preservative in vaccines) can be detected by the MELISA® test.

Avoidance of allergens often results in significant health improvement of the patient. Follow-up MELISA® testing usually shows reduced lymphocyte reactivity as well.

Diagnosis of active Lyme Borreliosis A recent development of the MELISA® test involves diagnosis of Lyme disease / active Borreliosis. Standard laboratory testing includes the serological test ELISA, which has very low specificity, and the Western blot, which is very specific. These tests can give false negative results in patients with early disease and therefore a misdiagnosis can occur.

MELISA® detects cellular immuno reactivity of active infections of Borrelia burgdorferi . The test improves laboratory diagnosis by confirming an active infection in patients with or without clinical symptoms.

Further applications MELISA® can also test for allergy to gluten, gliadin and casein, and testing to other food substances is being developed.

MELISA® test procedure

Blood in citrate tubes must arrive to the laboratory optimally within 24 hours after the blood has been drawn, maximally within 48 hours. White blood cells (lymphocytes) are isolated and tested against allergens chosen accordingly to the patient’s anamnesis and exposure to metals. A questionnaire, which goes through the most common sources of metal exposure, is available for download at www.melisa.org. The blood is incubated for five days and the lymphocyte reaction is measured by ways: one based on the uptake of radioisotope by dividing lymphocytes; the other by evaluation by microscopy. The level of reactivity is measured as a Stimulation Index (SI). A value over 3 indicates a positive reaction to a given allergen. The results are available in ten days.

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Licensing of the MELISA® test Certified laboratories may perform the MELISA® test under a license agreement. The MELISA® test is protected by trademark. Currently, laboratories performing the test are found Australia, Belgium, Germany, South Africa, Spain, Switzerland, United Kingdom and USA.

Laboratory equipment and material needed for MELISA® testing Cytocentrifuge

CO2 incubator

Table centrifuge for spinning of 50 ml falcon tubes

Betacounter for counting of radioactivity on filters

Harvester for DNA

Different multipipettes

Sterile bench (Laminoflow)

Microscope for light microscopy incl. objective 25x and 40x

Invert microscope (optional)

Water bath

The testing requires disposable material such as cell media, human serum, radioactive thymidine, plastic ware (pipettes, tubes, plates), metals solutions etc.

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Case study

Sue Collins was 10 years old when her first amalgam fillings were placed. She suffered from urticaria and rashes throughout her teenage years and suspected nickel allergy. As an adult, she underwent tubal ligation with a metal clip. She developed gallstones and had gall bladder removed. After placement of dental metal crowns, she experienced intense abdominal pains and was diagnosed with Irritable Bowel Syndrome. Additional crowns and bridgework were fitted and she experienced increasing muscle pain and weakness. Finally she received the diagnosis of fibromyalgia and chronic fatigue syndrome. The MELISA ® test showed a positive reaction to several metals in found in her dental restoration and metallic clips. After having the metals removed, her oral symptoms, IBS and fibromyalgia disappeared.

“I can honestly say I feel like a new person;

I have my life back.“

MELISA Diagnostics Limited

6 Heatherdene Mansions Phone: +44 (0)20 8133 5166 Cambridge Road Fax: +44 (0)20 8711 5958 Twickenham Skype: @ melisa.diagnostics TW1 2HR Email: info@melisa.org United Kingdom Web: www.melisa.or

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Reference literature Some articles are available from www.melisa.org/articles.php

Hypersensitivity reactions to metallic implants - diagnostic algorithm and suggested patch test series for clinical use. Schalock PC et al, Contact Dermatitis. 2012 Jan;66(1):4-19. Orthopaedic surgery in a patient with metal sensitivity. Adala R, Chakravarthy M, Srinivas V, Pai S. J Cutan Aesthet Surg. 2011 Jan-Apr; 4(1): 67-68. Metal allergens of growing significance: epidemiology, immunotoxicology, strategies for testing and prevention. Forte G, Petrucci F, Bocca B. Inflamm Allergy Drug Targets. 2008 Sep;7(3):145-62. The role of environmental factors in autoimmune thyroiditis.Hybenova M, Hrda P, Procházková J, Stejskal V, Sterzl I. Neuro Endocrinol Lett. 2010;31(3):283-9. LTT-MELISA® is clinically relevant for detecting and monitoring metal sensitivity. Valentine-Thon E, Müller K, Guzzi G, Kreisel S, Ohnsorge P, Sandkamp M. Neuro Endocrinol Lett 2006; 27(Suppl 1): 17-24 Hypersensitivity to titanium: Clinical and laboratory evidence. Müller K, Valentine-Thon E. Neuro Endocrinol Lett 2006; 27(Suppl 1): 31-35 Validity of MELISA® for metal sensitivity testing. Valentine-Thon E., et. al, Neuroendocrinology Letters, 2003;24(1/2):57–64 A novel lymphocyte transformation test (LTT-MELISA®) for Lyme borreliosis. Valentine-Thon E et al., Diagn Microbiol Infect Dis. 2006 Jul 27

Diagnosis and treatment of metal-induced side-effects. Stejskal V, Hudecek R, Stejskal J, Sterzl I. Neuro Endocrinol Lett 2006; 27(Suppl 1): 7-16 The role of metals in autoimmunity. Stejskal J, Stejskal V. Neuroendocrinology Letters 1999; 20:351-364 Metal-specific lymphocytes: biomarkers of sensitivity in man. Stejskal, V, et al. Neuroendocrinology Letters 1999; 20:289-298 Removal of dental amalgam and other metal alloys supported by antioxidant therapy alleviates symptoms and improves quality of life in patients with amalgam-associated ill health. Lindh U, Hudecek R, Danersund A, Eriksson S, Lindvall A. Neuro Endocrinol Lett. 2002;23(5-6):459-82. Nickel allergy is found in a majority of women with chronic fatigue syndrome and muscle pain - and may be triggered by cigarette smoke and dietary nickel intake. Regland B, Zachrisson O, Stejskal V, Gottfries CG. Journal of Chronic Fatigue Syndrome, Vol. 8(1) 2001 Mercury-Specific Lymphocytes: An indication of Mercury Allergy in Man. Stejskal V, Forsbeck M et al, Journal of Clinical Immunology, Vol. 16, No 1, 1996 MELISA® - an in vitro tool for the study of metal allergy. Stejskal V, Cederbrant K, Lindvall A, Forsbeck M, Toxicology In Vitro, 1994, vol 8, p. 991-1000