Development and initial validation of a modified lymphocyte transformation test (LTT) assay in patients with DRESS and AGEP

Table 1 Results of assessment of all proliferation assays tested as a potential detection method for LTT

ASSAY	Multiple time point read outs	Positive signal strength		Supernatant for cytokine analysis	Comments
ASSAY	Multiple time point read outs	PHA	Transact beads	Supernatant for cytokine analysis	Comments
BRDU	No, single	Neg SI [1] ANOVA (NS)	Weak (SI) 1.2 < ANOVA (NS)	Taken off before assay	Required spinning cells down to prior to fixation. Fixing step failed possibly due to T cells being small and non-adherent. Low sensitivity
CyQUANT™ NF	Yes	Weak SI < 1.2 ANOVA (NS)	Weak SI < 1.3 ANOVA (NS)	Taken off before assay	Required spinning cells down and removing supernatant. Signal weak after 60 min slight increase at 3 h
MTT	No, single	High SI 2.4 ANOVA *p < 0.05	High SI 2.6 ANOVA *p < 0.05	Taken off before assay	Assay sensitive enough but can only be read at one time point. Requires addition of DMSO for reading—thus no further analysis could be performed
XTT	Yes	High SI 2.8 ANOVA **p < 0.01	High SI 2.9 ANOVA **p < 0.01	Taken before or after assay	Simple addition of XTT Can be read at multiple time points (4, 6 and 24 h). Plates can be frozen after reading for further analysis

All future experiments were performed using XTT detection
NS Not significant, NEG Negative, BRDU bromodeoxyuridine, CyQUANT™ CyQUANT NF cell proliferation assay kit, MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, XTT tetrazolium salt (sodium 3´-[1- (phenylaminocarbonyl)- 3,4- tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate
SI greater > 2 positive, ANOVA *p < 0.05, **p < 0.01

ISSN: 1710-1492