Cytotest® - Cytotoxic Test®

Cytotest®- Cytotoxic test®

The Cytotest® - Cytotoxic test® is an in vitro diagnostic medical device (IVD) used for the identification and evaluation of food intolerances (non-IgE mediated adverse reactions to foods) through the observation and evaluation of any morphological alterations/modifications of the leukocytes present in the blood sample taken from the patient and placed in contact with food extracts.

The Cytotest®- Cytotoxic test® device is intended for professional use only.

Food intolerances

Food intolerances, not to be confused with IgE mediated food allergies, represent a concrete and very widespread phenomenon and are often responsible for the illnesses and conditions of discomfort that characterize our modern time.

Food intolerances do not involve the production of IgE antibodies but may produce delayed cell-mediated reactions to foods eaten daily and/or frequently.

Difference between allergies and intolerances

The term "allergy", i.e. "other reaction", was used for the first time in 1906 by Clemens Von Pirquet, a Viennese doctor, specialist in pediatrics.

He defined allergy as an altered, acquired and specific ability to react to substances foreign to the cells of the organism.

The reaction takes place through the action of the immune system, which represents a real defense system of the organism against all agents external to the organism itself; these foreign agents can be: harmful, such as microorganisms such as viruses, bacteria, etc., or harmless, such as, for example, chemical and food substances.

Very simply, the immune system is composed of different types of leukocytes such as lymphocytes, macrophages, neutrophilic granulocytes, monocytes, all called phagocytes, as they are responsible for the incorporation of toxic substances and their subsequent elimination; Lymphocytes are divided into two groups: T lymphocytes and B lymphocytes.

In turn, T lymphocytes are divided into: suppressor, auxiliary and killer.

B lymphocytes are very important for their ability to produce immunoglobulins, chemical substances directed against the foreign body, called specific antibodies, which can defend the body in various infectious and allergic diseases.

The immune response is divided into multiple aspects at the level of the various cellular components, both with direct intercellular contact and with the production of immunoglobulins.

When an infection with microorganisms occurs, the immune system usually retains the memory of the foreign body that caused the infection; thus, the phenomenon of immunity is constituted.

This complex phenomenon occurs through very specific stages in which, in short, some lymphocytes contact the foreign body (called antigen), pass information to other cells responsible for producing specific substances, called antibodies, which in turn attack the antigens.

Antibodies or immunoglobulins are of five main types: IgA, IgM, IgD, IgG (and subtypes) and IgE.

The defensive action, in addition to the production of immunoglobulins, occurs through the release of chemical mediators, such as, among the most important, histamine, serotonin, lysosomal enzymes, chemotactic factors, platelet aggregation factor.

There are four distinct types of defensive reactions of the immune system:

Type I

The reaction is mediated by IgE which is produced after exposure to allergens.

These immunoglobulins bind to the surface of mast cells, granulocytes and basophilic lymphocytes present at the tissue level.

When the allergen enters the body and there is contact with IgE, the cells will instantly release the aforementioned chemical mediators, first of all histamine, often responsible for allergic reactions in the strict sense: erythema, edema, itching, burning of the skin.

Type II

The reaction is mediated by IgM-IgG and cells, in this case killer lymphocytes.

In this reaction the organism fights living microorganisms by producing IgM and IgG immunoglobulins, which adhere to the host's cell wall so that killer lymphocytes can recognize it.

This type of reaction occurs, sometimes by mistake, even towards the very constituents of the organism; in this case we will have autoimmune diseases; the same phenomenon can be observed with the intake of toxic drugs.

Type III

The reaction is mediated by immune complexes. In this reaction, IgG and IgM immunoglobulins are formed against "soluble" antigens such as, for example, bacterial toxins, foods, chemical and natural substances.

The immunoglobulins produced join the antigens, activating the complement (series of proteins present in the serum), and this will attract the phagocytes which will destroy the foreign bodies.

Type IV

The reaction is mediated by T lymphocytes.

In this reaction, which usually develops two or three days after the appearance of the foreign substance, the cytotoxic T lymphocytes, which have been previously sensitized, attack the infected organic cells.

This type of reaction is what occurs in case of rejection of transplanted organs and degenerative diseases; It is also called delayed immunoreaction.

Since IgE antibodies were "discovered" and it was clear that many allergic symptoms (rhinitis, stuffy nose, conjunctivitis, watery eyes, asthma, etc.) are related to the quantity of these antibodies in the blood, only those where there is a high presence of the aforementioned antibodies, are called allergic diseases.

This was extremely important for defining the mechanisms underlying the various allergic processes but at the same time excluded from the definition of allergy all those phenomena of food intolerance which imply an involvement of the immune system but without the production of IgE antibodies.

We can talk about food allergies just in case of an excess of immunoglobulins E (IgE) in the blood which, in presence of foreign substance (allergen), be it pollen or dust or food, attach themselves to some types of white blood cells which release histamine which will cause inflammation, swelling of the tissues, etc.

Instead, we talk about food intolerances, when there is no production of IgE antibodies and the reactions are not immediate but chronic.

The disorders, in fact, are not directly related to the intake but can occur after some time, up to 72 hours later, and symptoms and diseases can develop in any organ system.

The mechanism that causes the triggering of these manifestations must be sought in the reaction of the immune system which, in the presence of certain foods, recognizes them as harmful and foreign, and consequently reacts.

Specifically, we talk about food intolerances when:

• there is no production of IgE antibodies;
• the reactions are not immediate but chronic; the disorders, in fact, are not directly related to the intake of the food but can occur up to 72 hours later and are a chronic reaction to foods consumed frequently such as wheat, milk, tomato, olive, coffee, etc.;

  often, since it is an accumulation of intolerated foods, it is precisely the most appreciated foods that are eaten daily that are responsible and the temporary feeling of well-being after taking them is felt because a mechanism similar to that of addiction is triggered from alcohol, drugs or tobacco;

  the phenomenon, therefore, can be accompanied by addiction disorders, dependence and relative abstinence in case of suspension;

• symptoms and diseases can develop in any organ system; the target can change over time and the disorders caused by food intolerances can also be very different from each other;
• the symptoms are not proportional to the quantity of the intolerated food introduced, therefore they are not dose-dependent: even small quantities can maintain the intolerance;
• cross reactions between foods of the same biological family or group are frequent; therefore, eating collateral foods means not detoxifying the body and maintain intolerance;
• after a period of avoidance of toxic foods or additives, intolerance disappears and foods can be gradually reintroduced.

Symptoms related to food intolerances

Sometimes, despite not being in the presence of a specific disease or pathology, recurrent and persistent disorders appear that cannot be solved, whatever the therapeutic approach.

The manifestations linked to food intolerances are sometimes related to severe stress conditions, which make the body more sensitive and which can lead to emotional disorders such as to weaken the psycho-physical balance even further.

The symptoms associated with food intolerances, which can also occur together, in a more or less important way, are:

asthenia, headache, nausea, meteorism, diarrhoea, swelling, post-prandial abdominal pain, recurrent infections, joint pain, skin changes such as urticaria, dyshidrosis, eczema, dermatitis, fluid retention, body weight disorders with variations both in excess and in defect but also chronic fatigue and insomnia and many others that often recognize food intolerance as the direct-indirect cause.

How to identify food intolerances

The Gold Standard in the diagnosis of food intolerance is the elimination diet which consists in eliminating for one or two weeks foods (the entire biological family) that are consumed by the patient most frequently, or which, based on the anamnesis, are suspected poorly tolerated.

The Cytotest® - Cytotoxic Test® is an alternative method to the elimination diet which is certainly quicker and more effective and is based on the observation and evaluation of any morphological alterations/modifications of the leukocytes present in the blood sample taken from the patient and placed in contact with food extracts.

Among the diagnostic methods, which have always been highly opposed and criticized, the Cytotest® or Cytotoxic Test® has constantly obtained recognition for its reliability and effectiveness.

History of technology

The awareness that foods could cause even serious disorders was already found in ancient times with the Greek doctor Hippocrates.

Over the centuries many other scholars have been able to verify the phenomenon but, only in 1924, the American doctors, Dr. H. Rinkel and, subsequently, Dr. T. G. Randolph enunciated their theories regarding the "anomalous" reactions that the intake of certain foods could cause in the individual.

The first to notice this correlation was Albert Rowe of Chicago in 1920.

Rowe noticed how numerous patients improved their various symptoms such as colitis, osteo-articular pain, asthenia, asthmatic bronchitis, rhinitis, hypertension, dysmenorrhea, psychiatric symptoms, headaches, dermatitis and more simply by suspending certain foods; in some cases, there was even complete remission of the symptoms previously complained of and resistant to the common therapies of the time.

The topic was addressed for the first time in 1951 by Dr. T. G. Randolph of Chicago who, based on the studies of Rowe and Rinkel, in the publication "Food Allergy", hypothesized how many chronic diseases could originate from eating disorders.

Randolph defined these facts as "Food Allergy" and this term could not help but prove incorrect just when Allergology was beginning to define and understand the mechanisms underlying "Allergies" and to consider as such only those manifestations linked to an involvement of the IgE and Histamine antibodies.

Certainly, Randolph was wrong in the definition but not in the clinical substance.

Therefore, it was clear and evident that all food phenomena, described and treated by Randolph, could not fall within the field of Allergology and this caused the distancing, skepticism and scientific marginalization of the American doctor and his students, even if by making hospitalized subjects fast under rigorous supervision and then re-feeding them with certain foods, he discovered a new therapeutic method.

However, this method was long and laborious and many weeks were needed to analyze around fifty foods.

It was therefore tried to find faster methods and at the same time reliable enough to achieve the same results as Randolph and then to understand if indicators could be found in the blood that demonstrated a food related cause of the patients’ complaints.

The first approaches date back to 1947 when some Anglo-Saxon immunologists, including Squier and Lee, observed in vitro a decrease in the number of leukocytes (up to a maximum of 33%) in patients who had been in contact with certain foods, but, the first study on the cytotoxic reaction of foods on the blood was conducted by Arthur Black who, in 1956, created the Cytotoxic Test.

Arthur Black observed the behavior of leucocytes in vitro when they were in contact with food allergens, and he detected cell morphological changes in foods that had been sensitive to individuals.

In presence of specific antibodies towards the allergen, the polymorphonuclear leukocytes presented toxic reactions with cell death that occurred within a period of between 15 minutes and a few hours.

If the reactions were strong and immediate, clinical allergen sensitivity was suspected.

Black's principle is the basis of numerous other research tests that have taken place over the years, especially in the USA and in Anglo-Saxon countries.

In 1959 one of the most famous immunologists, Prof. Byron Waksman, published several studies on the toxic effects of antigen-antibody reactions on cells and in particular the text "Cellular and humoral aspects in hypersensitivity conditions".

Further progress in the study and determination of an investigation method was achieved by various scholars, in particular by Bryan and Bryan, at the beginning of the 1960s.

The US test consisted of verifying in vitro the cytotoxic action of certain foods, an action that manifested itself with the morphological changes of the neutrophils contained in a blood sample and made it possible to diagnose food intolerances, avoiding patients' long and exhausting elimination diets.

In 1984 the founders of Cytodiagnostic srl imported the method from the United States and began to apply it in Italy with the primary objective of standardizing it, making it reproducible and reliable.

For over 15 years a team of engineers and professionals in the field has studied, designed and perfected the diagnostic kit until developing a robotic prototype to automate and standardize the production of the IVD which has been manufactured and commercialized in Italy and abroad by Cytodiagnostic srl since 2001.

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Material needed for the test

Carrying out the test requires instruments and materials commonly used in analysis laboratories.

To collect a blood sample:

• Skin disinfectants: alcohol, chlorhexidine, or povidone-iodine swabs or wipes
• Non-sterile gloves
• Tourniquet, disposable
• Needle system (needle and syringe, or needle and vacuum tube)
• Blood collection tubes (Tube with 0.5 ml of 3.8% sodium citrate or sodium citrate coagulation tube)
• Dressing materials (cotton wool, plasters, adhesive tape)

To perform the test:

• 1000-1500 rpm centrifuge with oscillating or rotating arm
• Non-sterile gloves
• 20-place laboratory slide tray
• 200 µl, 50 µl and 2 µl micropipettes
• Distilled water
• EPPENDORF cuvettes
• 18 x 18 coverslips
• Optical microscope with 40x objective

Preparation of the blood sample

Take an intravenous blood sample of between 2 and 5 ml;

The blood must be mixed in a test tube with 0.5 ml of 3.8% sodium citrate or the usual sodium citrate clotting test tube.

The resulting mixture must be centrifuged for 10 minutes at low speed (1000-1500 r/min) or left to sierate, preferably in the fridge, and in any case at a temperature between 4° and 8°C.

Test execution

The operations necessary for the use of the Cytotest® - Cytotoxic Test® device and the arrangements necessary for the success of the analysis, which the operator must strictly follow, are described in detail in the Instructions for use (IFU), present in each commercial package and in the user’s manual provided by Cytodiagnostic srl.

The reading shall start with observation of slide No. 00, negative control, which represents the comparative element (blank) for the detection of a positive reaction.

Positive reaction is only reported if the observation shows cell damage with a frequency above 60-70% within the same field and in the sum of the fields analyzed.

The reaction shall be classified according to the type of morphological change in the leucocyte.

1st Degree - NORMAL LEUKOCYTES:

• Normal packing of red blood cells;
• normochromic red blood cells;
• no morphological deformation of the red blood cells is observed;
• the leukocyte membrane is well preserved.

2nd Degree - SWOLLEN LEUKOCYTES:

• normal packing of red blood cells;
• normochromic red blood cells;
• vacuolated leukocytes with slight alteration of the membrane.

3rd Degree - VACUOLATED LEUKOCYTES:

• No packing of red blood cells;
• red blood cells tending to hypochromia;
• vacuolated leukocytes with partial membrane rupture followed by loss of cytoplasmic granules.

4h Degree - DISGREGATION OF LEUKOCYTES:

• the packing of red blood cells is becoming less and less evident;
• red blood cells appear hypochromic;

The leukocytes are broken up with a total rupture of the membrane

Test result

The test result shall include elimination of the food for a period depending on the degree of reaction, namely:

2° Degree = 4 months of abstinence

3° Degree = 6 months of abstinence

4° Degree = 6 months of abstinence

In addition, for each food found positive, the side foods belonging to the same biological family must be eliminated for two months.

It will be the nutritionist’s task to carefully evaluate the results in order to prescribe an appropriate diet, eliminating all foods that have been found to be biological families.