Table 3.

Validation results of analysis of regulatory T cell (Treg)-mediated suppression.

Validation part	Sample	Specificity	Result	Passed
Reproducibility
Coefficient of variation	2 + 1	≤5%	0.3	X yes □ no
	5 + 1	≤5%	1.0	X yes □ no
	10 + 1	≤5%	2.2	X yes □ no
	30 + 1	≤5%	4.5	X yes □ no
	50 + 1	≤10%	4.9	X yes □ no
Intermediate precision
Deviation from mean	2 + 1	≤±8%	5.0	X yes □ no
	5 + 1	≤±12%	7.2	X yes □ no
	10 + 1	≤±20%	12.5	X yes □ no
	30 + 1	≤±30%	−5.6	X yes □ no
	50 + 1	≤±40%	−39.8	X yes □ no
Coefficient of variation	2 + 1	≤10%	2.8	X yes □ no
	5 + 1	≤10%	3.8	X yes □ no
	10 + 1	≤10%	5.4	X yes □ no
	30 + 1	≤15%	4.8	X yes □ no
	50 + 1	≤15%	8.0	X yes □ no
Linearity
Coefficient of correlation in ascending range	100 + 1 to 5 + 1	≥0.8	0.908	X yes □ no
Range	All samples	≤10–≥90%	2.4–97.7%	X yes □ no
Accuracy
Retrieval rate	1 + 1	100 ± 5%	99.7%	X yes □ no
	5 + 1	100 ± 5%	98.4%	X yes □ no
	10 + 1	100 ± 10%	108.8%	X yes □ no
Limit of detection
QL proliferation		≤1%	0.4%	X yes □ no
QL suppression		≥95%	99.99%	X yes □ no

Responder cells and Treg were mixed at the indicated ratios of 2 + 1, 5 + 1, 10 + 1, 30 + 1, and 50 + 1. For reproducibility, triplicate analysis of responder cell to Treg ratios was statistically analyzed by coefficient of variation. The analysis of a different person on a different day was used to determine the intermediate precision. To determine linearity, 10 different responder cell to Treg ratios (1,000 + 1, 200 + 1, 100 + 1, 50 + 1, 30 + 1, 10 + 1, 5 + 1, 2 + 1, 1 + 1, and 1 + 2) were analyzed by correlation factor and linear regression. These results were compared with the theoretical real fraction contents to determine accuracy. The limits of detection were determined by analyzing six non-stimulated responder cell to Treg mixtures (1 + 1) and calculated with the formula: 10s/a (s indicating SD and a indicating slope of regression).

QL, quantification limit.