Validity and Reproducibility of Measurement of Islet Autoreactivity by T-Cell Assays in Subjects With Early Type 1 Diabetes

Validity and Reproducibility of Measurement of Islet Autoreactivity by T-Cell Assays in Subjects With Early Type 1 Diabetes Kevan C. Herold 1 , Barbara Brooks-Worrell 2 , Jerry Palmer 3 , H. Michael Dosch 4 , Mark Peakman 5 , Peter Gottlieb 6 , Helena Reijonen 7 , Sefina Arif 5 , Lisa M. Spain 8 , Clinton Thompson 1 , John M. Lachin 1 and the Type 1 Diabetes TrialNet Research Group * 1 Yale University, New Haven, Connecticut; 2 University of Washington, Seattle, Washington; 3 University of Toronto, Toronto, Canada; 4 Department of Immunobiology and the National Institute for Health Research Biomedical Research Centre at Guy's and St. Thomas' National Health Service Foundation Trust and Kings College, London, U.K.; 5 University of Colorado, Boulder, Colorado; 6 Benaroya Research Institute, Seattle, Washington; 7 National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland; 8 The George Washington University Biostatistics Center, Rockville, Maryland. Corresponding author: Kevan C. Herold, kevan.herold{at}yale.edu . Abstract OBJECTIVE Type 1 diabetes results from an immunemediated destruction of β-cells, likely to be mediated by T lymphocytes, but the sensitivity, specificity, and other measures of validity of existing assays for islet autoreactive T-cells are not well established. Such assays are vital for monitoring responses to interventions that may modulate disease progression. RESEARCH DESIGN AND METHODS We studied the ability of cellular assays to discriminate responses in patients with type 1 diabetes and normal control subjects in a randomized blinded study in the U.S. and U.K. We evaluated the reproducibility of these measurements overall and to individual analytes from repeat collections. RESULTS Responses in the cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays could differentiate patients from control subjects with odds ratios of 21.7, 3.44, and 3.36, respectively, with sensitivity and specificity as high as 74 and 88%. The class II tetramer and U.S. ELISPOT assays performed less well. Despite the significant association of the responses with type 1 diabetes, the reproducibility of the measured responses, both overall and individual analytes, was relatively low. Positive samples from normal control subjects (i.e., false positives) were generally isolated to single assays. CONCLUSIONS The cellular immunoblot, U.K.-ELISPOT, and T-cell proliferation assays can distinguish responses from patients with type 1 diabetes and healthy control subjects. The limited reproducibility of the measurements overall and of responses to individual analytes may reflect the difficulty in detection of low frequency of antigen-specific T-cells or variability in their appearance in peripheral blood. Footnotes *A complete list of co-investigators can be found in an online appendix. The TrialNet Study Group is presented at www.diabetestrialnet.org . Clinical trial reg. no. NCT 00212329, clinicaltrials.gov . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received February 19, 2009. Accepted July 16, 2009. © 2009 American Diabetes Association