Improving the timeframe between blood collection and interferon gamma release assay using T-Cell XtendÒ J.J.M. Bouwman a,*, S.F.T. Thijsen a, A.W. Bossink b
Department of Medical Microbiology and Immunology, Diakonessen Hospital Utrecht, The Netherlands Department of Pulmonology, Diakonessen Hospital, Utrecht, The Netherlands
Accepted 9 October 2011
T-Cell XtendÒ; TSPOT.TBÒ; Lithium heparin; IGRA; Interferon gamma; Tuberculosis
Summary Vacutainer CPT tubes require blood samples for TSPOT.TB to be processed within 8 h. In this study we evaluated the ability of T-Cell Xtend to maintain the number and function of lymphocytes after 24 and 48 h of blood storage, giving similar test results as in freshly isolated specimens. Methods: Whole blood specimens from 59 individuals were collected in Vacutainer CPT tubes (CPT) and lithium heparin (LH) tubes. CPT tubes were processed within 8 h. T-Cell Xtend was added to LH tubes after 24 or 48 h. We also left LH tubes untreated for 48 h. Total number of white blood cells (WBC) and proportions of lymphocytes and granulocytes were determined in the isolated Peripheral Blood Mononuclear Cells (PBMC). We also evaluated the performance of T-Cell Xtend in the TSPOT.TB assay. Results: PBMC yields from T-Cell Xtend treated LH samples did not differ from PBMC yields from CPT tubes, but T-Cell Xtend had a pronounced effect on the proportions of lymphocytes and granulocytes. The mean lymphocyte percentage in PBMCs isolated from fresh CPT blood was 84.31 Æ 1.14% (at t Z 48 h), but was decreased to 52.72 Æ 3.34% (p < 0.05) in untreated LH blood (at t Z 48 h). This effect was neutralized by T-Cell Xtend (85.44 Æ 0.74%). We observed a similar but opposite effect on granulocytes: The mean proportion in untreated LH blood was increased to 40.9 Æ 3.67% (p < 0.001) compared to CPT blood (8.26 Æ 0.89%). Treatment of LH samples with T-Cell Xtend (48 h) restored the proportion of granulocytes to 8.47 Æ 0.61%. Enumeration of spots in the TSPOT.TB assay demonstrated good agreement between CPT and T-Cell Xtend results, even after 48 h.
Abbreviations: PBMC, Peripheral Blood Mononuclear Cells; CPT, Cell Preparation Tube; LH, Lithium Heparin; LTBI, Latent Tuberculosis Infection; IGRA, Interferon Gamma Release Assay; MTB, Mycobacterium tuberculosis complex; PPD, Puriﬁed Protein Derivative; ESAT-6, Early Secreted Antigenic Target-6; CFP10, Culture Filtrate Protein 10; ELISA, Enzyme Linked Immuno Sorbent Assay; ELISPOT, Enzyme Linked Immuno Spot; IFN-g, Interferon Gamma; PHA, Phytohaemagglutinin; PMN, Polymorphonuclear leukocytes; SFC, Spot forming cells; TST, Tuberculin Skin Test; WBC, White Blood Cells. * Corresponding author. Department of Medical Microbiology and Immunology, Diakonessen Hospital Utrecht, Bosboomstraat 1, 3582 KE Utrecht, The Netherlands. Tel.: þ31 88 250 6075; fax: þ31 88 250 6695. E-mail address: email@example.com (J.J.M. Bouwman). 0163-4453/$36 ª 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jinf.2011.10.017 Please cite this article in press as: Bouwman JJM, et al., Improving the timeframe between blood collection and interferon gamma release assay using T-Cell XtendÒ, J Infect (2011), doi:10.1016/j.jinf.2011.10.017
J.J.M. Bouwman et al.
Conclusions: T-Cell Xtend efﬁciently removes granulocytes from PBMC suspensions and increases the proportion of lymphocytes. TSPOT.TB results from T-Cell Xtend treated blood samples are at least comparable to the results obtained from the current CPT method. Use of standard lithium heparin blood combined with T-Cell Xtend allows up to 48 h storage of blood samples for batched processing and may further decrease the rate of indeterminate TSPOT.TB results. ª 2011 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
At present, interferon...