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The introduction of second-generation QuantiFERON-TB (QFT) enables the diagnosis of Mycobacterium tuberculosis (TB) infection with high specificity and sensitivity. This in vitro diagnostic test uses 2 TB-specific proteins (ESAT-6 and CFP-10) to stimulate cells in heparinized whole blood and detects interferon-gamma (IFN-gamma) produced from blood cells. When QFT is done in laboratories outside of the hospital, several hours may be required to transport blood samples. We studied the relationship between QFT results and the time taken from collection of blood to incubation (preincubation time).Heparinized whole blood drawn from TB suspects was immediately transported to a laboratory. We started to incubate 4 aliquots of blood with ESAT-6, CFP-10, mitogen (phytohemagglutinin), and nil control, at 1, 3, 6, 9, and 12 h after blood was drawn. After incubation, the concentration of IFN-gamma in each plasma sample was determined by ELISA, and values E and C were expressed as the concentration of IFN-gamma with ESAT-6 or CFP-10, minus the concentration of IFN-gamma in the nil control. Value E or Cor =0.35IU/mL was considered positive,or =0.10 and0.35IU/mL as equivocal (gray zone), and0.10IU/mL as negative. We analyzed 8 patients with value E or Cor =0.10IU/mL at a preincubation time of 1 h.Value E and C decreased especially for preincubation time6 h. As a result, the interpretation of value E changed from "positive" to "equivocal" in 2 cases and from "equivocal" to "negative" in 2 cases. Interpretation of value C also changed from "positive" to "equivocal" or "negative" in 2 cases and from "equivocal "to "negative" in 1 case. Even if the higher of value E or C were used for analysis, QFT results changed in half of patients when preincubation time was6 h.Since QFT results in half of patients changed when preincubation time was6 h, incubation of whole blood should startor =6 h after blood drawing.