Abstract
We compared the performance of the T-SPOT.TB assay with blood used within 0 to 3.5 h after collection (control) to its performance with blood stored for 0 to 3.5, 5 to 8, 18 to 21, or 31 to 33 h with the addition of T-Cell Xtend (experimental), using samples from 154 participants. The 95.4% concordance between paired specimens indicated that blood can be stored for up to 33 h prior to T-SPOT.TB testing.
TEXT
It has been shown that blood samples should be as fresh as possible for the accurate analysis of cell-mediated immunity due to the rapid deterioration of cell function (4). Data on gamma interferon release assays (IGRAs) to assess latent tuberculosis (TB) infection have demonstrated that test performance can deteriorate within 24 h of phlebotomy for the T-SPOT.TB test (Oxford Immunotec Ltd., Abingdon, United Kingdom) (8) or within 6 h for the QuantiFERON TB (QFT) assay (Cellestis Ltd., Carnegie, Australia) (2, 3, 10). Therefore, commercial IGRAs have to be processed within 8 h (T-SPOT.TB test) or within 16 h (QFT-TB Gold In-Tube assay). Longer storage times would be advantageous as this would allow samples to be collected at any time during the day and shipped overnight to a testing facility. The objective of this study was to compare the performance of T-SPOT.TB using fresh blood samples stored for ≤3.5 h (control) to its performance using blood stored for up to 33 h (experimental) with the addition of T-Cell Xtend reagent (TCX) (Oxford Immunotec Ltd., Abingdon, United Kingdom).
Subjects with different TB risk factors were enrolled between February and May of 2011 at three clinical sites (The Ohio State University, in Columbus, Ohio; Hidalgo and Cameron County Chest Clinics, Brownsville, Texas; and The Ikhwezi Community Clinic in Cape Town, South Africa). The protocol was approved by the Institutional Review Board for each study site, and informed consent was obtained for each subject.
Blood was collected in lithium or sodium heparin Vacutainer tubes (Becton, Dickinson, NJ) and transported at room temperature. Three tubes of blood were collected from each subject, and the specimens were processed for the T-SPOT.TB assay in one of two study arms (Fig. 1). Controls were processed within 0 to 3.5 h in the absence of TCX. The second and third tube for each subject were processed as experimental tubes with TCX added. The experimental times were 0 to 3.5 h and 18 to 21 h for study arm 1 and 5 to 8 h and 30 to 33 h for study arm 2. The T-SPOT.TB assay and TCX were used according to the manufacturer's instructions, as previously described (11). Valid T-SPOT.TB assay results must have up to 10 spots in the Nil control and at least 20 spots in the positive control. The test is interpreted as positive when at least one of the antigens has ≥6 spots and negative when both antigens have ≤5 spots. The kappa statistic was used to measure the overall agreement between the paired test results.
Fig 1.
Study flow of patients and specimens. Times of storage before processing are indicated. a, One subject was excluded due to low cell count; b, two subjects were excluded, one due to >10 spots in the Nil control and the second due to low cell count; c, four subjects were excluded due to >10 spots in the Nil control. TCX, T-Cell Xtend reagent.
Among the 155 subjects enrolled, one was excluded due to low numbers of cells. Of the remaining study participants (n = 154), 62% were women, 34% white, and the mean age was 35 years (Table 1). Three specimens per subject yielded a total of 462 T-SPOT.TB assays and 308 paired results (Fig. 1). Six paired specimens were excluded due to invalid assay results: five had more than 10 spots in the nil control and one had insufficient numbers of cells at one of the follow-up time points. Final analysis was conducted on 154 subjects and 302 paired specimens. Each control was independently compared to the two corresponding samples processed with the TCX and with different storage times. Among the 302 possible pairwise comparisons, there was a total of 111 concordant positives, 177 concordant negatives, and 14 discordant results {95.4% [95% confidence interval (CI), 92.3% to 97.4%]; kappa = 0.90} (Table 2). Among the 14 discrepant pairs, six were within the borderline zone of the assay cutoff (5 to 7 spots). When the samples for each time point were analyzed separately, the agreement was between 93.2% and 98.6% with kappa values >0.85 for the four time brackets tested (Table 3). There was no significant difference between the T-SPOT.TB test results for each of the test times versus the control (P > 0.2).
Table 1.
Sociodemographic and medical conditions of study participants
| Characteristica | Value for group (n = 154)b |
|---|---|
| Mean age (yr) ± SD | 37 ± 12.7 |
| Male sex | 58 (38) |
| Ethnicity | |
| Non-Hispanic white | 52 (34) |
| Non-Hispanic black | 43 (28) |
| Hispanic | 51 (33) |
| Other | 5 (3) |
| Unknown | 3 (2) |
| BCG vaccination | |
| Yes | 39 (25) |
| No | 68 (44) |
| Unknown | 47 (31) |
| Self-reported medical conditions (not mutually exclusive) | |
| Asthma | 11 (7.1) |
| Diabetes | 9 (5.8) |
| Rheumatoid arthritis | 4 (2.5) |
| Steroid medication | 3 (1.9) |
| Methotrexate medication | 1 (0.6) |
| Lupus erythematosus | 1 (0.6) |
| HIV positive | 20 (13.0) |
| Active tuberculosis case | 4 (2.6) |
BCG, Bacillus Calmette-Guérin; HIV, human immunodeficiency virus.
Data are expressed as no. (%) unless otherwise indicated.
Table 2.
Agreement between the T-SPOT.TB assay results for the control samples and experimental samplesa
| Sample type | Result | No. of experimental samples (stored, with TCX) with indicated result |
Total no. | |
|---|---|---|---|---|
| Positive | Negative | |||
| Control (fresh, without TCX) | Positive | 111 | 8 | 119 |
| Negative | 6 | 177 | 183 | |
| Total | 117 | 185 | 302 | |
TCX reagent was added to specimens stored at room temperature after 0 to 3.5 h, 5 to 8 h, 18 to 21 h, and 30 to 33 h. All results were classified as positive if there were 6 spots or more and as negative if there were 5 spots or less.
Table 3.
Agreement between the T-SPOT.TB results for the control blood samples and the experimental blood samples after different storage times
| Experimental sample storage times (h) before addition of TCXa | Overall agreement [% (no. concordant/no. of paired samples); 95% CI] between results for control and experimental samples | Kappa value |
|---|---|---|
| 0–3.5 | 96.2 (75/78); 89.2–99.2 | 0.92 |
| 5–8 | 93.2 (69/74); 84.9–97.8 | 0.86 |
| 18–21 | 93.4 (71/76); 85.3–97.8 | 0.86 |
| 31–33 | 98.6 (73/74); 92.7–100 | 0.97 |
| All study results | 95.4 (288/302); 92.3–97.4 | 0.90 |
TCX, T-Cell Xtend.
The findings from this study indicate that the storage of blood at room temperature for up to 33 h does not affect the performance of the T-SPOT.TB assay in the presence of TCX. Concordance was high overall and consistent throughout all time ranges tested, indicating that storage time had no effect on the outcome of the assay.
No issues arising with the use of TCX were seen in the 44 (29%) subjects who had reported a medical condition which could affect their immune status (Table 1). A concordance of 100% was observed among the 24/44 non-HIV-infected subjects and 97.4% for the 20/44 subjects with HIV infection.
Though this study was limited to an adult population, the use of the TCX on stored blood has been reported previously for 11 children (age range 1.3 to 14 years), with concordant results for all subjects (11).
Activated granulocytes are responsible for inhibiting T cells, principally mediated by the release of H2O2 (6, 9), leading to reduced cytokine release by effector T cells (7). These effects may contribute to the observed reduced clinical sensitivity of both IGRAs when performed on stored blood without the removal of granulocytes (2, 3, 8, 10).
This study complements previous studies (1, 5, 11) showing that the TCX yields good results with extended storage times. Unlike Lenders et al. (5), who showed less confident results at 32 h, we had the highest agreement rate at the 31- to 33-h time point, with a kappa of 0.97. Unlike other published TCX studies, this study was performed with subjects with different TB risk factors that would be encountered in a clinical setting. We enrolled healthy participants, latent-TB-infected participants, recent contacts of TB patients, participants with suspected TB, and those diagnosed with active TB disease. In addition, this is the first report with information on clinical immunosuppression in subjects tested with the use of TCX. Lastly, this is the largest reported clinical study to date of paired clinical specimens comparing optimal fresh blood specimens processed at less than 3.5 h from blood draw to blood specimens with the addition of TCX processed at four longer time points.
In summary, the excellent agreement between the controls and each of the four time periods indicates that the use of TCX allows the storage of whole blood for up to 33 h without an impact on the results of the T-SPOT.TB test.
ACKNOWLEDGMENTS
This work was supported by Oxford Immunotec Ltd., United Kingdom, who contributed toward the costs of this study and to the provision of assay materials. S.-H.W. was supported by OSU Centers for Clinical Translational Science award number KL2 RR02574 from the National Centers for Research Resources.
We thank Ian Durrant and Jessica Pagan from Oxford Immunotec for their support, Julie Posey and Cassie Hines for assistance with recruitment and enrollment at OSU, Judy Hild for facilitating the use of the OSU Student Health Center for recruitment and enrollment at OSU, and Diana Gomez and Izelda Zarate for recruitment and enrollment in Brownsville.
Footnotes
Published ahead of print 18 April 2012
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