T-SPOT.TB (T-SPOT) is an interferon gamma release assay (IGRA) used to detect infection with Mycobacterium tuberculosis based on the number of spot-forming T cells; however, delays in sample processing have been shown to reduce the number of these spots that are detected following laboratory processing. Adding T-Cell Xtend (XT) into blood samples before processing reportedly extends the amount of time allowed between blood collection and processing up to 32 h. In this study, paired blood samples from 306 adolescents and adults at high risk for latent tuberculosis (TB) infection (LTBI) or progression to TB disease were divided into three groups: (i) early processing (∼4.5 h after collection) with and without XT, (ii) delayed processing (∼24 h after collection) with and without XT, and (iii) early processing without XT and delayed processing with XT. The participants' paired samples were processed at a local laboratory and agreement of qualitative and quantitative results was assessed. The addition of XT did not consistently increase or decrease the number of spots. In groups 1, 2, and 3, samples processed with XT had 13% (10/77), 28.0% (30/107), and 24.6% (30/122), respectively, more spots, while 33.8% (26/77), 26.2% (28/107), and 38.5% (47/122) had fewer spots than samples processed without XT. The findings suggest that XT does not reliably mitigate the loss of spot-forming T cells in samples with processing delay.