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

Abstract

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.

Susceptibility of A. fumigatus‐specific T‐cell assays to pre‐analytic blood storage and PBMC cryopreservation greatly depends on readout platform and analytes

“…Our findings confirm previous reports of granulocyte contamination of isolated PBMCs upon blood storage, demonstrably interfering with T‐cell metabolism and proliferation . Therefore, improvement of assay performance may be achieved by addition of antibodies cross‐linking granulocytes to erythrocytes and, thus, increasing the efficacy of granulocyte removal during PBMC isolation, in turn leading to improved T‐cell reactivity even after 24‐ to 32‐hour pre‐analytic storage . Other studies, however, found impaired assay performance even if blood was stored at conditions reducing granulocyte activation .…”

Tests for tuberculosis infection: landscape analysis

“…The laboratory procedure is more complex for T-SPOT.TB than QFT. The use of T-Cell Xtend reagents enables isolation of lymphocytes for up to 32 h (in contrast to 8 h without the reagents) after blood collection [18,19]. A new reagent kit, T-Cell Select, is claimed to extend the storage for up to 54 h before sample processing; however, we could not find published validation studies.…”

The performance of T‐cell Xtend reagent in increasing blood storage times for interferon gamma release assays

“…Similarly, as it was previously reported, this effect can be removed by treating blood with TCX before the start of the testing process.To our knowledge, five studies on TCX performance in T-SPOT.TB assay were published until present. Talbot et al17 reported a sensitivity of 96.8% after overnight blood storage. Lenders et al18 also showed that there was high agreement of T-SPOT.TB results between freshly isolated samples and 1 day delayed or 33 hours delayed blood samples, however, only 12 paired T-SPOT.TB results of 33 hours delayed samples were analyzed in that study.…”