Analytical Validation of Relative Average Telomere Length Measurement in a Clinical Laboratory Environment Marsha N. Blauwkamp, 1 Clare L. Fasching, 1 * Jue Lin, 1 Karl Guegler, 1 Evangelos Hytopoulos, 1 Drew Watson, 1 and Calvin B. Harley 1 Background: Average telomere length in whole blood has become a biomarker of aging, disease, and mortality risk across a broad range of clinical conditions. The most common method of telomere length measurement for large patient sample sets is based on quantitative PCR (qPCR). For laboratory-developed tests to be performed on clinical samples, they must undergo a rigorous analytical validation, currently regulated under CLIA. Methods: Whole blood samples from 40 donors were used in the analytical validation of methods for relative average telomere length (rATL) measurement. Three technical replicate DNA samples were extracted from each whole blood sample and placed in three independent wells on a sample plate. Each of these sample plates was assayed 12 times during the validation process. The study was conducted over a 20-day period, once in the morning and once in the evening, using 3 different operators. Results: Our process of rATL measurement beginning with DNA extraction followed by qPCR-based assay resulted in repeatability and reproducibility CV of <5% and amplification efficiencies near 100%. The validated assay was used to establish a reference interval derived from 2 cohorts of individuals: (a) San Francisco Bay area (n = 504) and (b) a US cross-sectional, demographic population (n = 357). Conclusions: We present advances in the establishment of a highly reproducible analytically validated process for determining rATLs in a CLIA laboratory environment. IMPACT STATEMENT Leukocyte telomere length is emerging as a biomarker for age-related disease risk. The challenge of comparing telomere length analyses across laboratories includes reconciling different methodologies, varied standardization, and high variability. Adoption of stringent controls and performance characteristics are central to pursuing clinical indications associated with small dynamic ranges of telomere lengths. We present the rigorous CLIA-inspired analytical validation of a relative average telomere length (rATL) measurement process, which we used to establish a normal reference interval. Given the growing number of associations between leukocyte telomere length and disease risk, particularly cardiac, increased consistency within and between assays benefits affected individuals. 1 Telomere Diagnostics, Inc., Menlo Park, CA. *Address correspondence to this author at: Telomere Diagnostics, Inc., 3603 Haven Ave., Suite A, Menlo Park, CA 94025. Fax 650-369-0644; e-mail cfasching@telomeredx.com. 3 Human genes: B2M, β-2-microglobulin; RPPH1, ribonuclease P RNA component H1. DOI: 10.1373/jalm.2016.022137 © 2017 American Association for Clinical Chemistry ARTICLES 4 JALM | 4 –16 | 02:01 | July 2017 ........................................................................................ Downloaded from https://academic.oup.com/jalm/article-abstract/2/1/4/5587480 by guest on 01 June 2020