The Stability of Never-Thawed Serum Biospecimens Stored in Vapor-Phase Liquid Nitrogen: Select Analytes from the National Health and Nutrition Examination Survey, 1999-2000 and 2005-2006

Introduction: The effect of prolonged storage (12-19 years) on selected laboratory test results is examined in the National Health and Nutrition Examination Survey biospecimen collection to determine whether biospecimens stored long term in vapor-phase liquid nitrogen provide valid results once remeasured.Methods: Biospecimens were selected for remeasurement using systematic random sampling for five analytes: cotinine, methylmalonic acid (MMA), vitamin A, vitamin E, and hepatitis C virus RNA (HCV-RNA). Measurements from the original specimens in 1999-2000 or 2005-2006 are compared with 2018-2019 measurements from the same survey participants and specimens. For quantitative analytes, measurement accuracy is assessed using standard method comparison procedures, precision is evaluated by comparing to quality control standards, and reproducibility is estimated by treating data like an incurred sample reanalysis. Qualitative measures are analyzed using concordance measures and exact binomial tests.Results: Observed proportional differences are 3%-12% for cotinine in people who do not smoke, 11% for cotinine in people who smoke, -8% to 1% for vitamin A, 8%-9% for vitamin E, and -6% to 8% for MMA. Precision estimates are within the standards established by quality control data and generally applicable quality goals. Differences between measurements are within 20% of the average value for at least 85% of all samples. For qualitative HCV-RNA and MMA results, we observe 99% concordance between measurements.Conclusions: Multipronged analysis showed that most differences are within acceptable ranges based on standard laboratory criteria for assessing accuracy, precision, and reproducibility. Results suggest future measurements and subsequent statistical analyses of stored serum specimens should be valid.