Abstract: T-SPOT.TB has been widely used to assay the Mycobacterium tuberculosis infection all around the world. As a new cell biological and immunological detection technology which its operation isn’t complex, T-SPOT.TB also raises a claim strictly on experimental condition, operation technique and quality control. The overall quality control including PBMCs separation, adjusting the number of PBMCs, cell incubation condition and result determination is the key steps to succeed as well as to be ignored. According to the test principle and clinical practice, we described and analyzed the different states of PBMCs separation including PBMCs separation, suspension preparation, incubation and result determination, especially, the error of PBMCs count affected experimental result. In addition, we established “one-step” and “calculation formula” method for PBMCs count according to traditional improved NiuBao leukocyte count plate and solution concentration dilution method, PBMCs count and standard cell suspension final concentration of 2.5×10⁵/100μl.

Key words: Tuberculosis/diagnosis, Interferon-gamma release tests, Leukocytes, mononuclear, Cell count, Quality control