An evaluation of the colorimetric assays based on enzymatic reactions used in the measurement of human natural cytotoxicity

Abstract

In recent years colorimetric assays based on an enzymatic reaction such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay have been used in an attempt to replace the conventional isotopic assay for cell-mediated cytotoxicity. To clarify the problems in the colorimetric assays for natural cytotoxicity, K562 cells were employed as target cells and peripheral mononuclear cells (PBMCs) from cancer patients were used as effector cells. No correlation was found between the 51Cr assay and the MTT assay ( P>0.05) or the N-acetyl-beta- d-glycosaminidase (NAG) release assay ( P>0.05) in 16 cancer patients. Labeling effector cells showed that the 51Cr release levels of such cells in 19 chemotherapy patients were significantly higher than the levels from target cells in this group ( P<0.01) and from effector cells in the control group ( P<0.01). There was no correlation between the positive and negative 51Cr assays ( P>0.05). The sensitivity of the MTT assay was greatly decreased by washing K562 cells prior to loading MTT solution. Enzyme release occurs as a result of cell metabolism and elevated enzyme release is associated with freezing. These findings indicate that the colorimetric assays based on an enzymatic reaction are not suitable for the detection of natural cytotoxicity in all populations, and are especially not suitable for the assay of natural cytotoxicity in chemotherapy patients.