Table 1.

Validation results of analysis by flow cytometry.

Validation part	Parameter	Acceptance	Result		Passed
Reproducibility	Coefficient of variation	≤5%	0.3	2.0%	X yes □ no
			0.5	2.5%
			0.7	0.8%
Intermediate precision	Deviation of mean	≤10%	0.3	8.7%	X yes □ no
			0.5	4.9%
			0.7	7.1%
	Coefficient of variation	≤8%	0.3	5.6%	X yes □ no
			0.5	3.8%
			0.7	4.0%
Linearity	Correlation	≥0.9	1,000		X yes □ no
	Linear regression	n.a.	y = 0.973x + 0.002		X yes □ no
Range	Cell fraction	0.04–1	0.04–1		X yes □ no
Accuracy	Deviation from actual value	0.3 ± 0.03	0.3	0.022	X yes □ no
		0.5 ± 0.05	0.5	0.019
		0.7 ± 0.07	0.7	0.014
	Recovery	100 ± 10%	0.3	92.8%	X yes □ no
			0.5	96.1%
			0.7	98.1%
Limit of detection	Quantification limit (QL) (10,000 cells)	≤0.04	0.016		X yes □ no
Limit of detection CD8	QL (300,000 cells)	≤0.001	0.001		X yes □ no
Specificity	Fluorescence in channel 1 or 2	Meets specificity	Meets specificity for all antibodies tested		X yes □ no
Efficiency of antibodies	Fraction of positive cells	Within range of development	All antibodies within range		X yes □ no

Positive cells and negative cells were mixed at the indicated fractions 0.3, 0.5, and 0.7 to represent 30, 50, and 70% of the final cell mixture. For reproducibility, triplicate analysis of three different fractions of positive cells 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 fractions of positive cells 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 negative cell fractions and calculated with the formula: 10s/a (s indicating SD and a indicating slope of regression). Antibody specificity and efficiency were determined by using positive and negative cell fractions, respectively.