Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania

Background. T-SPOT.TB is an interferon gamma release assay for detecting Mycobacterium tuberculosis infection. The requirement to process within 8 hours is constraining, deters use, and leads to invalid results. Addition of T Cell Xtend reagent may allow delayed processing, but has not been extensively field tested. Design. Consecutive AFB smear positive adult tuberculosis patients were prospectively recruited in Dar es Salaam, Tanzania. Patients provided a medical history, 1–3 sputum samples for culture and 1 blood sample which was transported to the laboratory under temperature-controlled conditions. After overnight storage, 25？μL of T Cell Xtend reagent was added per mL of blood, and the sample was tested using T-SPOT.TB. Results. 143 patients were enrolled: 57 patients were excluded because temperature control was not maintained, 19 patients were excluded due to red blood cell contamination, and one did not provide a sputum sample for culture. Among 66 evaluable patients, overall agreement between T-SPOT.TB and culture was 95.4% (95%CI; 87.1–99.0%) with Kappa value 0.548. Sensitivity of T-SPOT.TB when using T Cell Xtend reagent was 96.8% (95%CI; 88.8–99.6%). Conclusions. When T Cell Xtend reagent is added to specimens held overnight at recommended temperatures, T-SPOT.TB is as sensitive as the standard assay in patients with tuberculosis. 1. Introduction The T-SPOT.TB (Oxford Immunotec Ltd, Abingdon, UK) is an interferon gamma release assay (IGRA) for detection of latent Mycobacterium tuberculosis infection (LTBI). This assay detects T-lymphocytes specific for M. tuberculosis antigens that are absent from M. bovis BCG and most environmental mycobacteria [1]. Currently, T-SPOT.TB must be performed within 8 hours of sample collection. Logistically, this requires that patients have blood drawn early enough to transport the sample to the laboratory, which must assay samples on the day of receipt. This precludes batching samples, which would save resources and testing supplies. Moreover, in many countries, laboratories are centralized and are not in close proximity to where the blood sample is drawn. Therefore, it is not possible to transport samples to arrive at the receiving laboratory for processing on the same day. To allow greater test processing flexibility, the manufacturer of T-SPOT.TB developed a proprietary reagent called T Cell Xtend, which they report can be added to whole blood samples stored at 10–25°C to increase this timeframe up to 32 hours [2]. 2. Materials and Methods The ethical review boards of Dartmouth Medical School (Hanover NH),