Method for determining compatibility between donor and recipient using flow cytometric detection of alloreactive T cells

摘要

A method for determining compatibility between a donor and a recipient using flow cytometric detection of alloreactive T cells, obtained from the following steps: a) a donor / recipient pair to be analyzed A step of adding a label to one blood sample, hereinafter referred to as labeled sample, which is suitable for distinguishing from a sample derived from the recipient and is the donor / recipient to be analyzed. Calling the other blood sample obtained from the pair below the unlabeled sample, b) diluting both samples at least once with the first medium and separating the cellular components of the sample from the liquid phase; c) Re-incorporating cell components, particularly blood sample residues of labeled samples and blood sample residues of unlabeled samples, of the same volume, separately into the second medium. Step d) mixing a part from the unlabeled sample and a part of the labeled sample, in particular in a ratio of approximately 1: 1 to obtain a mixture, e) described in step d) of claim 1 , Stimulating antigen presenting cells (APC) in a mixture of labeled and unlabeled samples by addition of anti-CD28 antibody, or anti-CD28 antibody and anti-CD49d antibody, followed by f) 35-39 ° C., A step of performing a first incubation of the mixture, preferably at a temperature of 37 ° C. for 1.5 hours to 2.5 hours, preferably 2 hours, in a protective gas atmosphere or CO 2 atmosphere, g) secretion inhibition Adding the agent, h) mixing the sample from step d) of claim 1, i) followed by a period of at least 2.5 hours at a temperature of 35-39 ° C., preferably 37 ° C. , Protective gas A step of performing a second incubation, j) optionally lysing red blood cells, k) CD4 lymphocytes or CD8, eg, with labeled anti-CD4 antibody and / or anti-CD8 antibody A step of detecting lymphocytes by flow cytometry using lymphocyte labeling and flow cytometry detection; l) lymphocytes activated by intracellular cytokines and lymphocytes expressing an activation marker are flow sites Detection step by measurement, m) the following results in flow cytometry detection: a. Label positive / CD4 positive / CD69 positive / INF-γ positive, b. Label negative / CD4 positive / CD69 positive / INF-γ positive, c. Label positive / CD8 positive / CD69 positive / INF-γ positive, d. A method comprising the step of identifying the presence of alloreactivity between an individual from which a labeled sample is obtained and an individual from which an unlabeled sample is obtained when label negative / CD8 positive / CD69 positive / INF-γ positive appears.