EP2010909A2

EP2010909A2 - Method of analysis in blood for identifying food products which are potentially dangerous for individuals susceptible to develop reactions towards some food products

Info

Publication number: EP2010909A2
Application number: EP07734652A
Authority: EP
Inventors: Gilles Costongs
Original assignee: Immogenics Ltd
Current assignee: Immogenics Ltd
Priority date: 2006-02-28
Filing date: 2007-02-28
Publication date: 2009-01-07
Also published as: WO2007099454A2; WO2007099454A3; FR2897942A1

Abstract

The invention concerns a novel blood analyzing method. Said method consists in: (i) calculating the average of a certain number of blood parameters from 5 unused samples; (ii) comparing individually each of the samples comprising a food, for one of said parameters, with the average of the unused sample of the same subject; (iii) calculating the difference between the unused sample and the sample comprising a food; (iv) multiplying said difference by a weighting coefficient; (v) adding said differences. The test performed according to the method enables the foods to be avoided by the subject to be determined for a more or less long period of time, leading to long term weight loss, thereby providing a sensation of well-being to the subject.

Description

METHOD OF BLOOD ANALYSIS

The present invention relates to a blood test method for selectively identifying potentially hazardous foods in individuals susceptible to develop reactions to certain foods.

These reactions to foods can cause including obesity, and more generally a feeling of ill-being in these subjects.

The method according to the invention is particularly well suited when these dangerous foods can not be easily identified; this method is not suitable for otherwise healthy individuals with serious anaphylactic reactions each time they ingest a particular food, or for children with potential food susceptibility, who are fed with a food substitute. cow's milk to the exclusion of all other foods, which is both appropriate and a financially sound diagnostic approach,

It is widely known that food is digested and fragmented before being absorbed for use in producing energy; the human body does not recognize food in a more complex form and then considers it as a foreign body against which it launches an immune response in order to fight it.

The physiological process of digestion transforms food into a form that can be used by cells primarily in glucose and free fatty acids. Glucose is released from dietary carbohydrates such as starch or sucrose by hydrolysis into the interior of the body. small intestine, then absorbed into the blood where cells can metabolize glucose into an energetic form.

However, subjects may react to certain foods with an immune response. If this is the case, the glucose can not be metabolized but is converted into glycogen. The fats will remain in the form of fats or will be oxidized as acetylated acids. Such immune responses to certain foods induce a state of chronic inflammation and revolve around small undigested food particles.

Macromolecular aggregates (small food particles), which have already undergone partial digestion, reach the small intestine where they will essentially be further digested into smaller particles through pancreatic enzymes and bile. These molecules of smaller dimensions (including water, electrolytes, glucose, amino acids and fatty acids) are then absorbed into the body through three mechanisms: through the intestinal mucosa where they are carried away by the blood circulation through small blood vessels, through the membrane Plasma epithelial cells of the intestine also referred to as enterocytes (a process commonly referred to as the "transcellular" route) and ultimately through narrow junctions between enterocytes (a process usually referred to as the "paracellular" route). However, small amounts of macromolecular aggregates will inevitably escape total digestion. When this phenomenon occurs, these aggregates will become antigenic and thus generate an immune response. This immune response starts in the gut but spreads rapidly when these aggregates are found in the bloodstream. In order to better understand the purpose of the invention, it is essential to specify how the chemical processes of the cells of the human body interact. The cell is the place of the body where hundreds of thousands of reactions occur. The two processes of particular interest in weight and energy are the immune process and the metabolic process. Both of these processes are regulated by a receptor on the cell surface that can activate insulin receptors. The immune receptor (integrin) will trigger an immune response and the metabolic receptor (tyrosine kinase) will trigger increased production of energy.

In order to better understand how these metabolic and immune processes interact, the cell should be seen as having a door that allows the food to return and then produce energy through the metabolic process. If the cell door is open, the food can enter the cell and will be used to efficiently produce energy. However, if this door is closed, the food is blocked outside the cell and can not be converted into energy: it will instead be stored as grease; tyrosine kinase is responsible for this opening of the gate but also for the reactivation / stimulation of intracellular glycogen / lipid metabolism for cell growth. These two immune and metabolic processes are regulated by a chemical agent free IgF1 ("insulin-like growth factor 1"). The levels of IgF1 in the body are limited and are produced by the liver for more than 80%. L ³ IgF1 is able to bind to both integrin (immune system) and tyrosine kinase (metabolism). Of course, if more IgF1 is needed or used by one of these receptors, there will be less for the other.

Several conditions are necessary for the cell door to be opened, in particular a balanced supply of available foods as well as IgF1. free, likely to bind to tyrosine kinase (metabolism). It is important to know what happens when the body reacts to the food that is being absorbed.

When the human body has an immune response to a particular food, several phenomena occur. Once spotted the foreign food, signals are sent to the cells that launch the immune response, then implementing a large amount of free IgF1 that will bind to the integrin (activator of the immune system) to order the cells to fight the intruder. This operation will leave less free IgF1 able to bind to tyrosine kinase (metabolism), the key that opens the cell door and activates, as has already been explained, glycogen / lipid metabolism and cell growth. As a result, foods will have less ability to enter the cells, but will tend to stay outdoors as fat. Another consequence of the above is that the body's muscles will have less energy and will send a signal to the brain that more food is needed to get more energy. However, eating more will not solve the problem because the subject will be able to absorb food that will cause a reaction in his body. If this is the case, no matter how much food is absorbed, energy will not be supplied to the muscles and fatigue and hunger will persist.

These general principles having been recalled, there are many subjects likely to develop reactions to food for which there is the possibility of reaction to at least one food and for which the possibility of pathogenic mechanisms other than only IgE also exists. Some of these suspect foods can be identified by a detailed historical study, the use of diets and questionnaires, or even the use of skin tests; however, these approaches are of limited value. An object of the present invention is therefore to present a method applicable to a specific food panel, likely to identify foods that can trigger pathogenic reactions, also identifying foods that do not lead to such reactions (we will then in the presence of negative results).

The method according to the present invention will thus result in a selection of foods likely to be included or excluded from a food program as follows:

1. foods that can be absorbed;

2. foods that can not be absorbed for a certain number of weeks, or even beyond one year. In order that the present invention is perfectly understood, the description of the method according to the invention will now be presented.

30 ml of blood is usually taken from the subject; the blood will then be anticoagulated with K2EDTA or K3EDTA provided that the blood sample reaches the testing laboratory within 8 hours of collection. If this delay is not respected, the blood will be anticoagulated with citrate or preferably with a mixture citrate / EDTA or SACD or other equivalent substance. The samples will then be stored at a constant temperature, preferably of the order of 4 ° C.

The method according to the invention will be made even more precise if a preliminary screening is carried out on the complete blood sample before the test of the method according to the invention is carried out. Such a screening will concern at least one of the six parameters and preferentially to all these parameters:

1. If the rate of leukocytes alive is less than 0.75 (less than 75% of leukocytes are alive) the test will not be performed and a new sample will be requested from the subject.

2. If nucleated red blood cells are present in the subject's complete blood sample, this sample will be analyzed a second time; if, during this second analysis, nucleated red blood cells are still present, it will be necessary to determine whether the sample actually contains nucleated red blood cells, or whether the identification of nucleated red blood cells is due to the aging of the sample

(non-viable lymphocytes): a. in case of nucleated red cells actually present, the complete blood sample may be refused; b. in the case of nucleated red blood cells present (with more than 10 nucleated red blood cells / 100 leucocytes) due to an aging of the sample, a new sampling will be required.

3. If anemia is present, ie for the following values: hemoglobin level for a female subject less than 6 mmol / 1, hemoglobin level for a male subject less than 7 mmol / 1, average blood volume less than 80 fl (iron type) or greater than 110 fi (type vitamin B6, B12, folic acid), the test will not be performed.

4. If the blood sample indicates inflammation (abnormal differentiation: neutrophil count greater than 8 / nl, lymphocyte count greater than 4 / nl), the test may be refused and the subject may eventually be subjected to an inflammatory

5. If there is leukopenia (leukocyte count below 2 / nl, leukocytosis greater than 12 / nl), the test may be refused and the subject may eventually be subjected to an inflammatory protocol.

6. If there is platelet aggregation (platelet aggregation alert threshold achieved), the subject will need to provide a new blood sample.

When the sample has passed the various stages of the preliminary screening, if this has taken place, it can then be transferred to a Tecan pipetting robot for dilution with food fragments; the Tecan robot will preferably perform a dilution of 80 .mu.l of blood for 120 .mu.l of food extract, ie a ratio of 40/60.

After pipetting, the mixture can be incubated at room temperature for at least 10 minutes and at most for 45 minutes; after incubation, the blood / food extract mixture will be tested according to the invention using a Cell-Dyn Sapphire or Cell-Dyn 4000 automaton from Abbott Laboratories.

Foods used for the preparation of food extracts are obtained according to the Allergon or Sigma method or any other equivalent method and diluted with a 0.2% phenol saline solution; they are mixed in a rotary machine for 36 hours, centrifuged at 1200 g and filtered on a 20 μ Sartorius filter. The solution thus obtained may be aged for 7 days and stored at 4 ° C .; before use this solution will be diluted 1: 5. After dilution, these dietary extracts thus diluted are stable for 6 days.

Since the preliminary screening has been successfully carried out and the food extracts are ready, the method according to the invention can be started using preferably a Cell-Dyn Sapphire or Cell-Dyn 4000 automaton.

Abbott Laboratories which is a hematology analyzer using a combination of 4 techniques (with laser technology) to determine the immune response of a given subject to tested foods; each food is tested separately. At the end of this test, more than 4 million living cells will have been tested on an individual basis to determine whether they give rise to a reaction to these foods. Among these cells, it is the leucocytes that will be studied.

The software that is associated with the Cell-Dyn analyzer, or whatever, will take into consideration the following different categories of cells: - neutrophils - monocytes

- eosinophils

- lymphocytes

Once the test of the method according to the invention has been carried out, these cell categories will appear in the form of separate clouds in a dispersion diagram which will be interpreted in order to determine the food program that will be proposed to each subject.

The algorithm that is used in this method consists of two streams of data:

- Com Port data,

- Datalog extraction data.

Thanks to the Com Port data, it is the stability of the Cell-Dyn analyzer, the Tecan robot and the quality of the sample that will be controlled; these controls will involve the use of the following parameters: red blood cell impedance, hemoglobin, mean red blood cell volume, red blood cell dispersion spectrum and disability alert data. The red blood cell impedance and hemoglobin are selected for their analytical stability. , including in old samples. Mean cell volume and dispersion spectrum of red blood cells are chosen because these parameters, in addition to being stable for old samples, are independent of the dilution ratio.

The acceptance for the interval of the numerical data is 0.6 x complete blood sample +/- 10% (5% for the average cell volume and for the dispersion spectrum of the red blood cells); if the acceptance criteria are rejected three times in a row, the analyzer must be stopped and can not be restarted until the problem has been resolved. The acceptance interval for the disability alert data is as follows: If disability data is shown three times in a row, stop the sample analysis and troubleshoot the system.

With regard to the extraction data, the average of the following parameters (viable fraction of leucocytes, leucocytes, mean cell volume, dispersion spectrum of red blood cells, neutrophil 0 °, neutrophil 7 °, neutrophil 90 °, neutrophil 90 ° depolarized, red fluorescence 3, 0 ° lymphocyte, 7 ° lymphocyte, 7 ° optical map, 90 ° optical map, 0 ° lymphocyte variation coefficient, 7 ° neutrophil variation coefficient, 90 ° neutrophil variation coefficient, 90 ° depolarized neutrophil coefficient of variation , red fluorescence coefficient of variation 3, 0 ° lymphocyte coefficient of variation, 7 ° lymphocyte variation coefficient, 7 ° optical wafer coefficient of variation, 90 ° optical wafer coefficient of variation) must be calculated individually from 5 blank samples. If a blank sample differs by more than 5%, it must be rejected and will not be included in the calculation; no more than two blank samples can be rejected (if these blank samples show an abnormal dispersion of the results, they will be subject to manual verification).

Each sample, along with the food extract, should be compared individually for each of the above parameters with the average obtained with the virgin sample of the same subject. The difference between the average of the sample with food extract and the average of the blank sample will be multiplied by a weighting coefficient; the sum of all these differences is ranked decreasingly. The weighting coefficient is between 0 and 100 depending on the viable fraction of leucocytes which itself must be between 75 and 100. When the sum of said differences is less than a value between 50 and 100, the food does not will not be forbidden.

As previously mentioned, the food program will result in two groups of plans, namely:

1. Foods that can be absorbed, 2. Foods that can not be absorbed for several weeks or more than one year.

The foregoing is determined in the Cell-Dyn analyzer, or the like, by examining the changes in living cells made by food relative to the state of these living cells in the reference samples (blood tested in salt dilution). According to a preferred embodiment of the present invention, the leucocytes analyzed will be neutrophils; the aforementioned scatter plots will thus separate the neutrophils into:

1. normal neutrophils: no reaction

2. neutrophils with toxin: presence of a toxic reaction 3. neutrophils with phagocytosis: presence of a toxic reaction with phagocytosis

4. neutrophils with degranulation: presence of a toxic reaction with degranulation.

5. dead neutrophils. With regard to the foods to be avoided for 5 weeks, a toxic reaction as mentioned above is common; phagocytosis, degranulation or death of neutrophils are rare. When switching to foods to be avoided for 10 weeks, a toxic reaction with phagocytosis and degranulation is common. Neutrophil death and degranulation are common for foods that can never be absorbed again.

The test according to the method of the invention will be repeated for each food; in order to obtain a broad spectrum of foods intended to integrate into one of the four above-mentioned categories, the order of 115 different foods and 5 reference samples will preferably be tested, thus resulting in a suitable food program for the subject. The latter will have his own program and this program may be periodically adapted to take into account the normal variations in the life of the subject.

The thresholds of the Cell-Dyn analyzer are quite flexible, which means that old samples (up to 72 hours) can be validly tested. Since each immune response is absolutely unique, it means that if a cell reacts to one of the substrates (foods) presented to it, this reaction itself can be measured. The test performed according to the method of the present invention is sophisticated enough that each subject receives detailed and accurate results. The blood of the subject will thus determine the foods giving rise to a positive reaction with respect to the samples tested. The method according to the present invention presents a certain improvement over the usual methods insofar as it is adapted to each subject by providing a list of foods to be selectively absorbed (without restriction, excluded for a limited period or forever) ). The dietary program determined by this method will avoid the disadvantages of traditional diets in which lipids and / or carbohydrates are regularly excluded; in this type of diet, after a while, the message from the cells is that the fuel is not suitable (no lipids or no carbohydrates), resulting in the closing of the door of the cell with the previously described consequences. In this case, we traditionally speak of the "yoyo" effect having the following characteristics: - once all the nutrients have been used, the cell closes its door and falls into apoptosis, thus increasing the level of IgF1; - V apoptosis in turn gives a signal to the brain in order to decrease the level of IgF1 produced; - At the end of the apoptosis phase of the cell, the IgF1 level will decrease and the newly absorbed nutrients will be transformed into adipose tissue.

Such a "yoyo" effect has dramatic consequences: - the lost weight will come essentially from the muscle mass,

at the end of the apoptosis phase mentioned above, all the lost muscle tissue will be replaced by adipose tissue.

As a result, the exclusion of lipids and / or carbohydrates will result in weight loss, only in the short term, followed by an increase in weight causing a feeling of frustration of the subject who will have lost his impression of well-being that he had provided her initial weight loss.

The method according to the invention and the resulting food program will avoid this feeling of frustration by favoring a long-term weight loss.

Claims

1) Method of blood analysis which consists in: a) introducing a food extract into a blood sample of a subject, b) testing the effect of said extract on the leucocytes, c) distributing said cells after the test in different categories corresponding to a food program specific to the subject.

2) A method according to claim 1 wherein said leukocytes are divided into: a) normal b) with toxin c) with phagocytosis d) with degranulation e) dead

3) A method according to one of claims 1 or 2 wherein the diet program will be: f) no restriction on food when the leukocytes will be essentially normal, g) foods should not be absorbed for a number of weeks when the leucocytes will be with toxin, with phagocytosis, with degranulation or dead. 4) Method according to claim 3 characterized in that said food program will not absorb the foods involved for a minimum period of 5 weeks when said leukocytes will be essentially with toxin or phagocytosis.

5) Method according to any one of claims 1 to 4 characterized in that said leukocytes are neutrophils.

6) Method according to any one of claims 1 to 5 characterized in that the blood sample has been treated with an anticoagulant prior to the implementation of said method.

7) Method according to any one of claims 1 to 6 characterized in that a preliminary screening is performed on the blood sample before the implementation of the test according to the invention.

8) Method according to claim 7 characterized in that said preliminary screening comprises at least one of the following parameters: at. viable fraction of leucocytes less than 0.75 b. presence of nucleated red blood cells in the blood sample c. presence of anemia d. presence of inflammation e. presence of leucopenia f. presence of platelet aggregation.

9) Method according to any one of claims 1 to 8 characterized in that said blood sample is diluted at a rate of 40% blood and 60% of food extract. 10) Method according to any one of claims 1 to 9 characterized in that said food extract is diluted in a salt solution of phenol 0.2%.

11) Method according to any one of claims 1 to 10 characterized in that it comprises the following steps: i. calculate the average of the following parameters (viable fraction of leucocytes, leucocytes, average cell volume, red cell dispersion spectrum, 0 ° neutrophil, 7 ° neutrophil, 90 ° neutrophil, 90 ° depolarized neutrophil, 3 red fluorescence, 0 ° lymphocyte, lymphocyte 7 °, optical wafer 7 °, optical wafer 90 °, coefficient of variation lymphocyte 0 °, coefficient of variation neutrophil 7 °, coefficient of variation neutrophil 90 °, coefficient of variation 90 ° neutrophilic depolarized, coefficient of variation fluorescence red 3, coefficient 0 ° lymphocyte variation, 7 ° lymphocyte variation coefficient, 7 ° optical wafer variation coefficient, 90 ° optical wafer coefficient of variation) from 5 blank samples, ii. individually compare each of the samples containing a food, for each of the above parameters, to the average of the blank sample for the same subject, iii. calculate the difference between the blank sample and the food sample, iv. multiplying said difference by a weighting coefficient, v. add the said differences.