Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

Table 5 IFNγ real time RT-PCR LOQ and LOD

Expected copies	Detected copies (Mean, n = 12)	SD	Number of positive results/total 12 tests	% Recovery	%CV
IFNγ			12/12
100,000	104,508	15,676	12/12	105%	15%
10,000	9,032	1,174	12/12	90.3%	13%
1,000	942	198	12/12	94.2%	21%
100	80	27.2	12/12	80%	34%
10	10	NA	8/12	NA	NA
1	1	NA	3/12	NA	NA
CD8
100,000	93,334	8,400	12/12	93%	9%
10,000	10,533	2,001	12/12	105%	19%
1,000	1,035	134	12/12	103%	13%
100	109	41.4	12/12	109%	38%
10	14	NA	10/12	NA	NA
1	2	NA	3/12	NA	NA

NA, not applicable (spiked copy number at 10 and 1 did not show detection in all 12 tests, therefore SD, % Recovery, and %CV is not analyzed).
IFNγ plasmid at different copy numbers were used for assessment of LOQ and LOD. Expected and detected values are shown. % Recovery (Detected/Expected) and % CV (SD/Mean) are calculated to assess assay accuracy and precision, respectively.

ISSN: 1479-5876