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

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) ass ay 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 Cr-51 assay and the MT T 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 51 Cr release levels of such cells in 19 chemotherapy patients were significan tly higher than the levels from target cells in this group (P<0.01) and fro m effector cells in the control group (P<0.01). There was no correlation be tween the positive and negative Cr-51 assays (P>0.05). The sensitivity of t he MTT assay was greatly decreased by washing K562 cells prior to loading M TT solution. Enzyme release occurs as a result of cell metabolism and eleva ted enzyme release is associated with freezing. These findings indicate tha t the colorimetric assays based on an enzymatic reaction are not suitable f or the detection of natural cytotoxicity in all populations, and are especi ally not suitable for the assay of natural cytotoxicity in chemotherapy pat ients. (C) 2001 Elsevier Science B.V. All rights reserved.