Variation in T-SPOT.TB Spot Interpretation between Independent Observers from Different Laboratories

T-SPOT.TB is a specific assay for the diagnosis of tuberculosis. The assay needs to be performed with freshly isolated cells, and interpretation requires training. T-SPOT.TB has been used in various clinical-epidemiological settings, but so far no studies have evaluated the effect of interobserver variation in test reading. Our aim was to evaluate variation between different observers in reading T-SPOT.TB results. The study was nested within an ongoing cohort study, in which part of the T-SPOT.TB had been performed with frozen material. Culture plates were read visually by four different observers from two laboratories and by two automated readers. Of 313 T-SPOT.TB assays, 235 were performed with fresh cells and 78 were performed with frozen cells. No significant difference was found between results obtained with fresh cells and those obtained with frozen cells. The percentage of positive results varied between readers by maximally 15%; five/six raters were within a 6% difference in positive results. Analysis of the observed interrater differences showed that some individuals systematically counted more spots than others did. Because test interpretation includes subtraction of background values, this systematic variance had little influence on interindividual differences. The test result as positive or negative varied between independent raters, mainly due to samples with values around the cutoff. This warrants further study regarding determinants affecting the reading of T-SPOT.TB.Roughly a century after the introduction of the tuberculin skin test (TST), the recent development of gamma interferon release assays (IGRA) for specific detection of infection with Mycobacterium tuberculosis has realized a new class of immunodiagnostic tests that have extensively been evaluated for detection both of active tuberculosis (TB) and of latent TB infection (1, 2, 4, 7). T-SPOT.TB and QuantiFERON-TB Gold in-tube are the commercially available and approved IGRA formats, being based on culture of isolated peripheral blood mononuclear cells (PBMCs) and of whole blood, respectively. Numerous studies that evaluated the use of IGRA have been published in the past several years, showing their particular value for detection of latent TB infection in populations with high rates of false-positive TSTs due to Mycobacterium bovis BCG vaccination or exposure to nontuberculous mycobacteria (3, 5). T-SPOT.TB is based on the enzyme-linked immunospot assay technique in which cells responding with gamma interferon production after antigen stimulation are visualized as spots, which must be enumerated. This can be done by use of an automated spot reader or by using a magnifying glass. The assay is performed in four wells with different stimulations: medium as a negative control, phytohemagglutinin as a positive control, and peptides of the TB-specific antigens ESAT-6 (panel A of the assay) and CFP-10 (panel B of the assay). One of the disadvantages of T-SPOT.TB is that it must be performed with fresh material, which may not always be convenient. As the assay is based on single-well culture for each stimulus, random variability cannot be detected. Another disadvantage is that counting the spots might lead to variation when results are read by different observers or automated readers.Thus far, no studies have addressed the interobserver variability of the T-SPOT.TB. In the present study, these issues were addressed by using material obtained within an ongoing cohort study in The Netherlands in which part of the T-SPOT.TB assay was performed with frozen material for logistical reasons (blood arriving in the laboratory on a Friday was frozen since the assay needed to be completed 20 h later). We evaluated the reading of the T-SPOT.TB plates in two laboratories by different observers and by two automated readers. Next, we compared results of T-SPOT.TB obtained with freshly isolated cells to those obtained with frozen and thawed cells.