title: Improving the timeframe between blood collection and interferon gamma release assay using T-Cell Xtend®.
abstract: 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 ± 1.14% (at t = 48 h), but was decreased to 52.72 ± 3.34% (p < 0.05) in untreated LH blood (at t = 48 h). This effect was neutralized by T-Cell Xtend (85.44 ± 0.74%). We observed a similar but opposite effect on granulocytes: The mean proportion in untreated LH blood was increased to 40.9 ± 3.67% (p < 0.001) compared to CPT blood (8.26 ± 0.89%). Treatment of LH samples with T-Cell Xtend (48 h) restored the proportion of granulocytes to 8.47 ± 0.61%. Enumeration of spots in the TSPOT.TB assay demonstrated good agreement between CPT and T-Cell Xtend results, even after 48 h.
  
  
  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.
authors: Department of Medical Microbiology and Immunology, Diakonessen Hospital Utrecht, The Netherlands. jbouwman@diakhuis.nl
authors: J J M
authors: JJ
authors: Bouwman
authors: S F T
authors: SF
authors: Thijsen
authors: A W
authors: AW
authors: Bossink
mesh_terms: D001800:Blood Specimen Collection; D006098:Granulocytes / Q000502:physiology; D006493:Heparin / Q000097:blood; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000097:blood; D059425:Interferon-gamma Release Tests; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000502:physiology; D018655:Lymphocyte Count; D008214:Lymphocytes / Q000502:physiology; D011933:Reagent Kits, Diagnostic
chemical_list: D011933:Reagent Kits, Diagnostic; D007371:Interferon-gamma; D006493:Heparin