The test is based on the analysis of the reaction leukocytes have when placed in contact with aliment extracts chosen to assay the effect of intolerance. Survey is directly accomplished on leukocyte cells through optical microscope.

Protocol followed in the test execution is that proposed by Bryan (1960 and 1967) and consisting of six steps:

• 5 ml of blood are collected in a anticoagulant-containing tube (sodium citrate: 3.8 g/L) and centrifuged at 1500- 2000 x g for 20 minutes.

Two phases will form: a lower sector, containing red cells, and a higher zone composed by plasma. At the interface it is possible to see a thin white layer where leukocytes are laid.

• By a disposable Pasteur pipette, 0.5 ml of material are harvested from this interface, sucking leukocytes the more as possible, but below and above phases too.
• Collected material is transferred in a tube, already containing 2.5 ml of water, and it is gently mixed with it through the pipette.
• Single drops of this diluted material are put in contact with aliment extracts pre-settled on slides.
• Slides are consequently covered with proper cover-slide and let in incubation for 10- 15 minutes.
• Slides are then analysed under optical microscope with 400 times as a final enlargement.

By observing leukocytes, it is possible to attribute different degrees of reaction to the food extract analysed according to the following scale:

These different reaction levels are defined on the basis of a leukocyte state scale which starts from a normal condition, passes through a swelling one, a vacuolization, to end with a breaking situation. They are likewise established on the relative amount of leukocytes which have reacted.

Before proceeding in the analysis of intolerance reactions towards the various aliments, a control slide is assayed. Here, leukocytes are placed in contact with the only components which are used in the extraction from the aliments. This phase is performed in order to ensure that subsequently tested reactions are specific and relative to the aliment, and also to check whether the amount of white cells in the microscopic optical field to be analysed is the right one (work procedural control). This moment allows, besides, to check the health state of the cells of the specific patient.