Cytotoxic Method

HISTORY OF THE CYTOTOXIC METHOD

Among the diagnostic methods for food intolerances, often opposed and criticized, the Cytotest method has been the most appreciated for research and reliability.
The modification of leukocytes caused by the reaction antigen-antibody has been subject of several studies and it was observed under different aspects.

In 1947 some important Anglo-Saxon immunologists, Squier and Lee between the others, observed in vitro a decrease of the number of leukocytes (until a maximum of 33%) in allergic patients after being in contact with food reagents.

 In 1956 Arthur Black, during his studies, determined that the alteration of the leukocytes indicates an allergic reaction; he observed the modifications of leukocytes in vitro as a reaction to allergens toxic for some sensitised patients.

If in contact with an allergen, leukocytes showed toxic reactions with the cells’ expiry, in a lapse of time from 15 minutes to few hours.

In 1959 one of the most well-known immunologists, Byron Waksman, published different studies about the toxic effects of the reaction antigenantibody on the cells and specially the text “Cellular and humoral aspects in hypersensitivity conditions”.

In the sixties, thanks to the studies of different researchers (Bryan and Bryan between them), there were further progresses in the study and standardisation of this method of research.
Bryan and Bryan codified this methodology making it easier, reliable and repeatable.

REQUIRED MATERIALS

Test-tube containing 0.5 ml of sodium citrate 3.8%
5 ml syringe with a 22G needle
Centrifuge (speed 1000 to 2000 RPM), with swinging or rotating arm
Micropipette of 200 μl 50 μl and 2 μl
Distilled water
Cuvette EPPENDORF
Cover slides 18 x 18 mm
Optical microscope with 40x objective

CYTOTOXIC METHOD

The method used to diagnosticate food intolerances is based on the leukocytes modifications in addition of the dried food allergens.

Patients with positive results to one or more substances are suggested to eliminate these foods from their diet, for a period of time depending on the grade reaction, in order to detoxicate the human body: in this period of time white blood cells “forget” that that food is toxic for the body.

Food intolerances are not perennial; after a period of abstinence, usually foods can be reintroduced in the diet, avoiding eating them too often and to cause a new accumulation of toxins in the body.

Here follow some important considerations about food intolerances:

• They’re a chronic reaction to foods which are assumed too often (wheat, milk, tomato, olive, coffee);
• The diseases caused by food intolerance do not appear immediately after the assumption of the foods, but can appear even after 72 hours;
• Diseases and symptoms can involve any organ, apparatus, system;
• Food intolerances can coexist with addiction disturbs, dependence and abstinence syndrome in case of suspending;
• Symptoms are not directly proportionate to the quantity of the toxic food introduced; they do not depend on the dosage and even small quantity of substances can cause intolerance;
• Frequently there are cross-reactions between substances members of the same biological group, so during the abstinence from a toxic food, patients must avoid also collateral foods, or they will not detoxicate the body;
• Sometimes food intolerances can be caused by modifications of the immune system (granulocites neutrofili, Ig 4 – interleukin 1) due to stressful life, chemical substances and pollution.

PREPARING THE SAMPLE

Results of Cytotest® are not conditioned by the assumption of some food before having the blood test.

Patients are advised not to take cortisone in the preceding 10 days before the exam.

Antihistaminic and other groups of medicines do not alter the results of the test.

We take from 2 to 5 ml of intravenous blood and we mix it in a test-tube with 0.5 cc of sodium citrate 3.8%.

If the quantity of blood taken is lower than 2 ml, we recommend reducing the sodium citrate to 0.25 cc.

The mixture can be centrifuged for 10 minutes at a low speed (from 1000 to 2000 RPM) or left in a refrigerator, at a temperature from 4 to 8 C° (do not freeze it).

The blood sample must be analysed within 72 hours.

READING THROUGH THE MICROSCOPE

The operator has to adopt the following important advices during the execution of the test.

Arrange the slides (starting from the n° 0 negative check) on the special tray, paying attention to put them with the label on the left side and the identification number on the top.

Read from the left to the right.

Each reading needs the observation of more fields for each substance (4 or 5).

We can speak of a positive reaction only if the reading shows a cells damage with a frequency of more than 60%-70%, either in the same field or in the sum of all the examined fields.

If we find cells damage with a higher frequency in all the analyzed fields, and a reaction to all the substances composing the kit, we can suppose that:

•  The blood sample has been taken more than 72 hours before the reading;
• The assemblage of the sample has not been made in the proper way.

In this case, test result is not reliable.

Positive reactions are classified in classes, based on the morphological alteration of the leukocytes.

Here follow the 4 grades of reaction:

GRADE 1: NORMAL LEUKOCYTES

• No changes to the leukocytes’ structure and Red Blood Cells (RBC) stacked
• RBC normo chromic
• No morphological changes of red blood cells
• The cells’ membrane is undamaged

GRADE 2: SWOLLEN LEUKOCYTES

• RBC stacked
• RBC normo chromic
• Vacuoles and leukocytes with a light alteration of the membrane

GRADE 3: VACUOLES IN LEUKOCYTES

• No stacking of the RBC
• RBC are hypo chromic
• Vacuoles in leukocytes with a partial breaking of the membrane, followed from the loss of the cytoplasmatic granules.

GRADE 4: DISINTEGRATION OF THE LEUKOCYTES

• No stacking of the RBC
• RBC are hypo chromic
• Disintegration of leukocytes, with the total breaking of the membrane

BIOLOGICAL GROUPS

Patients with positive results to one or more substances are suggested to eliminate them ompletely from their diet, for a period of time depending on the degree of reaction.

The purpose of the elimination is to detoxify the human body: it helps white blood cells to “lose memory” that a food is toxic for the patient.

Patient must eliminate also foods belonging to the same biological family or foods containing similar substances, in order to avoid a crossreaction.

Food intolerances are not perennial; usually after a period of abstinence, the toxic foods can be introduced again in the diet, but avoiding to eat them daily, because they could cause a new

accumulation of toxins in the body.

The following schedule reassumes the most important biological groups:

REPORT CYTOTEST®

Cytodiagnostic Ltd. provides free of charge to users of the Diagnostic Kit software for Cytotest report.

The software allows a medical report in the following languages: Italian, English, Spanish, Romanian.

DVANTAGES AND DISADVANTAGES OF CYTOTEST®

The use of Cytotest® in the diagnosis of food intolerances allows to make use of several advantages, here summarised:

• it’s a test in vitro, so there is any risk for the patient;
• it’s rapid;
• results are not altered by the gravity or the variety of intolerances;
• it’s very sensitive and it can point out even the slightest intolerances;
• it’s economical if compared to other techniques;
• it’s very selective and precise and it can give at the same time the positiveness for one - two - three foods.

We can summarise Cytotest® disadvantages in three basic points:

• the preparation of the slides of the kit is long-lasting and complicated;
• it can be made only with alive cells and the samples of blood must be used in a quite short time (about 72 hours);
• the reading of cell’s reactions is subjective; it depends on the laboratory accuracy and on the researcher’s skills. This is the reason why the Cytodiagnostic s.r.l. supplies the diagnostic kit for the execution of the test only after an intensive training of a day, practising the reading of 10 tests at least, and it gives continuous advice in the reading technique and interpretation of results