Pre-analytical and analytical variability associated with the interferon gamma release assays
Tuberculosis (TB) is a leading cause of death worldwide despite the availability of drug treatments. Prevention in low-incidence settings such as the United States (U.S.) focuses on identification and treatment of TB infection (TBI). The TSPOT.TB assay (TSPOT) and the QuantiFERON-TB Gold In-Tube assay (QFT-G) are interferon-γ release assays (IGRA) that aid in the diagnosis of TBI. The study’s objective was to investigate the pre-analytical and analytical variability associated with the IGRA assays. Three specific aims were used to address this issue. (1) Determine the agreement between the TSPOT assays results read by an Elispot reader compared to assays read by trained technicians. Automated and manual TSPOT results among U.S. HCWs were compared. Cohen’s kappa coefficient was used to determine the inter-rater agreement. Univariate and multiple logistic regression were used to investigate selected variable contributions. No prognostic factors were associated with agreement of TSPOT results between counting methods. The inter-rater agreement for all test results was good (kappa=0.71), so commercial Elispot readers and manual counts were found to have good agreement of TSPOT results in a low TB burden setting. With no TBI gold standard, it cannot be determined if one TSPOT reading method is better than another. (2) Compare the distributions and potential risk factors associated with the positivity of TSPOT assays due to the ESAT-6 and CFP-10 antigens in the TSPOT test that has the antigens in separate wells. There were 576 positive results in 8805 TSPOT assays representing 2418 participants. A significant difference was detected in the number of positive TSPOT results due to > 8 spots over the nil in either ESAT-6 well, CFP-10 well or both antigen wells at baseline over a 12 month (M) ( p<0.001) period, but not for the 18M follow-up. Among our HCW samples with positive TSPOT assays, a larger proportion of positive TSPOT results showed reactivity to CFP-10 (49.5%) than ESAT-6 (19.6%). (3) Identify the key hospital medical units having high indeterminate QFT test results, and reduce the proportion of indeterminate results by conducting an education program targeting nurses within problematic units. An online link to a pre-test survey, education presentation, and post-test survey was emailed out to 332 nurses in 9 units chosen for their high proportion of indeterminate QFT-G tests. Of the 332 nurses emailed, 37 completed both tests within the time allotted. The nurses that completed the education program had a significantly higher post-education test score than on the pre-test (55.3% versus 72.5%, p < 0.001). The targeted education program was able to successfully increase nurses’ knowledge of blood collection and handling procedures for the QFT-G test, but it remains to be seen if the increased knowledge translates to a real world reduction in the rate of indeterminate QFT-G test results at Houston Methodist Hospital (HMH).^
Agarwal, Saroochi, "Pre-analytical and analytical variability associated with the interferon gamma release assays" (2016). Texas Medical Center Dissertations (via ProQuest). AAI10248624.