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IN VITRO METHOD AND KIT FOR ASSESSING CELL IMMUNITY
WO19180295
This invention consist in a vitro method to evaluate the cellular immunity of animals or humans from a sample of biological fluid (saliva, whole blood or whole blood previously treated with anticoagulant). A sample of mononuclear cells is extracted, which are suspended in a saline solution that is subsequently frozen. Previously, the mononuclear cells present in this saline solution must be quantified. Freezing takes place at -20°C for one hour and has the function of subjecting the mononuclear cells to stress so that they release analytes into the environment. The procedure continues with the thawing of the mononuclear cell saline and its centrifugation. The supernatant liquid resulting from the centrifugation is then collected and the content of analytes or biomarkers that are indicators of inflammation and/or oxidative stress in the collected supernatant liquid is determined. Finally, the content of these analytes is compared with the average reference content of the same analytes obtained by replicating this method under the same conditions from at least two samples of the same type of biological fluid with mononuclear cells from animals or from healthy humans. The evaluation of the biomarkers can be determined in an absolute or relative way. The values of each biomarker per mononuclear cell in the sample are therefore estimated, in particular the values of each biomarker per lymphocyte and the absolute values of this biomarker throughout the de sample. For the application of this method, a new kit has been designed that, from a sample of biological fluid, evaluates cellular immunity in animals or humans in a simple, fast and reliable way
This method of cellular immunity evaluation and the kit developed for its application present the following advantages, with respect to the main techniques currently used for the same purpose: - It is a lower cost procedure since it does not require the use of specific equipment such as flow cytometers. - It requires less time to obtain results since it does not require incubation, cell culture or the addition of antigens. - It does not require specialized personnel for its application. - This is a very sensitive test. The assessment of biomarkers or analytes can be determined both absolutely and relatively, which makes the test more sensitive than if it were expressed only in absolute values, since relative values measure the cellular immune response capacity of each cell. - It is a flexible method for determining a more or less extensive set of analytes, and allows the determination of the analytes in the supernatants collected in step (vi) of the in vitro method of the invention just after performing such step (vi) or later, and it is also possible to transport them for the determination of the analytes in other laboratories. - This method has a high stability, so once thawed, samples are stable for up to 24 hours and can be sent to laboratories for further analysis.