METHODS AND SYSTEM FOR VALIDATING MEASUREMENT RESULTS OBTAINED BY FLOW CYTOMETRY Russian patent published in 2024 - IPC G01N15/14 

FIELD: physics.

SUBSTANCE: invention relates to methods of performing validation for assessing reliability and reproducibility of measurement results. Method for validating measurements using an analytical instrument based on measuring fluorescence with respect to a population of coloured target cells in a test sample involves: (a) negative staining in a first portion of a reference sample containing target cells expressing a target cell marker, where the negative staining provides for the introduction into the first part of the reference sample of a fluorescent dye to exclude dead cells, a non-specific antibody conjugated with a fluorochrome, and a specific antibody capable of specifically binding a target marker on a target cell; (b) positive staining in the second part of the reference sample, where the positive staining in the second part of the reference sample in (b) includes the introduction into the second part of the reference sample of a fluorescent dye eliminating dead cells, and specific antibody conjugated with fluorochrome; (c) analysing the first portion and the second portion of the reference sample using the instrument to obtain a fluorescence measurement result indicative of the concentration of target cells; (d) based on the results obtained in (c), diluting the positively stained target cells in the second part of the reference sample with negatively stained target cells in a first part of a reference sample to obtain a diluted sample having the highest concentration of target cells, from which a series of dilutions is obtained, where each diluted sample is characterized by a nominal concentration of target cells, each nominal concentration of cells exceeds the concentration of target cells, which is indicated by the fluorescence measurement result of the negatively coloured first part in (a), where the nominal concentration in each diluted sample differs from the nominal concentration in each of the remaining diluted samples, and where the highest concentration of target cells in the dilution sample is 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05% or 0.02%; (e) analysing a series of diluted samples from (d) using an instrument to obtain a series of fluorescence measurement results; (f) comparing the nominal cell concentration from (d) and measuring the fluorescence from (e) for each diluted sample to quantify the effectiveness of the staining method using the instrument, where the comparison in (f) involves performing a statistical calculation of the difference between the nominal cell concentration and the fluorescence measurement for each diluted sample with the determination of at least one of the linearity, the range, accuracy, precision, limit of detection (LOD) and lower limit of quantification (LLOQ) for the instrument and method of staining, and where determination of LLOQ involves identification of concentration of target cells, associated with a predetermined precision criterion and a predetermined accuracy criterion.

EFFECT: improved methods of validating analytical methods and systems.

17 cl, 9 dwg, 14 tbl