Impact of blood volume, tube shaking, and incubation time on reproducibility of QuantiFERON-TB gold in-tube assay

Abstract

Gamma interferon (IFN-γ) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-γ TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-γ response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Agvigorous minus nilgentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.

Fig 1

Schematic overview of the study design. Subjects underwent QuantiFERON-TB Gold In-Tube (QFT-GIT) testing to investigate the effects of blood volume variability, shaking variability, and incubation duration variability on results. Ag, antigen.

Fig 2

Effect of blood volume variability on QuantiFERON-TB Gold In-Tube (QFT-GIT) results. (A) Distribution of blood volumes drawn into nil and TB Ag tubes for 30 subjects. The horizontal line represents the median volume. Ag, antigen; CV, coefficient of variation. (B and C) IFN-γ TB Ag − nil values for 33 uninfected (B) and 17 infected (C) subjects tested with the indicated blood volumes in the TB Ag tubes. The assay cutoff value for positive results (TB Ag − nil value of ≥0.35 IU/ml) is marked with dashed lines. Values of <0 IU/ml are shown as 0 IU/ml. The Wilcoxon signed-rank test was used to compare differences in medians.

Fig 3

Effect of tube shaking on QuantiFERON-TB Gold In-Tube results. (A and B) IFN-γ values in nil (A) and TB Ag (B) tubes with gentle and vigorous shaking for 40 subjects (23 uninfected and 17 infected). (C and D) IFN-γ TB Ag − nil values with gentle and vigorous shaking. (C) Responses for gently shaken nil and TB Ag tubes (TBAg_Gen - Nil_Gen) versus those for vigorously shaken nil and TB Ag tubes (TBAg_Vig - Nil_Vig). (D) Responses for gently shaken nil and TB Ag tubes versus those for gently shaken nil and vigorously shaken TB Ag tubes (TBAg_Vig - Nil_Gen). The assay cutoff value for positive results (TB Ag − nil value of ≥0.35 IU/ml) is marked with dashed lines. Values of <0 IU/ml are shown as 0 IU/ml. The Wilcoxon signed-rank test was used to compare differences in medians. Ag, antigen; Gen, gentle; Vig, vigorous.

Fig 4

Effects of incubation duration variability on QFT-GIT results. IFN-γ TB Ag − nil values for 33 uninfected (A) and 17 infected (B) subjects tested with the indicated durations of incubation are shown. The cutoff value for positive results (TB Ag − nil value of ≥0.35 IU/ml) is marked with dashed lines. Values of <0 IU/ml are shown as 0 IU/ml. The Wilcoxon signed-rank test was used to compare differences in medians.