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<p>Serum samples are widely used in metabolomics studies and are typically stored at −80&#xa0;°C after collection. Thawing of frozen serum prior to sample preparation is therefore an unavoidable pre-analytical step that has the potential to influence metabolomic data quality and biomarker discovery. Previous studies have reported inconsistent effects of thawing, largely reflecting differences in analytical platforms, metabolome coverage, quantification accuracy, sample size, and study design. Direct assessment of the impact of thawing on biomarker discovery remains rare. Here, we systematically evaluated the effects of three commonly used serum thawing protocols—room-temperature thawing, thawing at 4&#xa0;°C, and thawing in an ice bath—on serum metabolomic profiles and biomarker discovery using high-performance chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS). Serum samples from 95 healthy individuals (54 males and 41 females) were analyzed under each condition. Statistically significant and reproducible differences were observed among the three thawing methods for a small subset of metabolites, demonstrating that thawing conditions do exert measurable effects on metabolite levels. However, these effects were modest relative to intrinsic biological variation. Supervised modeling and other statistical analyses showed that biologically meaningful patterns, including sex-dependent metabolic separation, were consistently preserved across all thawing conditions. These findings demonstrate that while the thawing method can introduce detectable metabolite-level differences, it does not materially compromise biomarker discovery performance when a single protocol is applied consistently within a study. Thus, any of the three thawing methods can be used in practice, provided consistency is maintained, with the choice guided primarily by logistical considerations.</p>

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Comparative Evaluation of Three Serum Thawing Methods and Their Effects on Metabolome Integrity and Biomarker Discovery

  • 期刊论文

  • 发表日期：

    2026年5月16日

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  • Deying Chen,
  • Guanghua Ma,
  • Lanjuan Li,
  • Liang Li

摘要

Serum samples are widely used in metabolomics studies and are typically stored at −80 °C after collection. Thawing of frozen serum prior to sample preparation is therefore an unavoidable pre-analytical step that has the potential to influence metabolomic data quality and biomarker discovery. Previous studies have reported inconsistent effects of thawing, largely reflecting differences in analytical platforms, metabolome coverage, quantification accuracy, sample size, and study design. Direct assessment of the impact of thawing on biomarker discovery remains rare. Here, we systematically evaluated the effects of three commonly used serum thawing protocols—room-temperature thawing, thawing at 4 °C, and thawing in an ice bath—on serum metabolomic profiles and biomarker discovery using high-performance chemical isotope labeling (CIL) liquid chromatography–mass spectrometry (LC–MS). Serum samples from 95 healthy individuals (54 males and 41 females) were analyzed under each condition. Statistically significant and reproducible differences were observed among the three thawing methods for a small subset of metabolites, demonstrating that thawing conditions do exert measurable effects on metabolite levels. However, these effects were modest relative to intrinsic biological variation. Supervised modeling and other statistical analyses showed that biologically meaningful patterns, including sex-dependent metabolic separation, were consistently preserved across all thawing conditions. These findings demonstrate that while the thawing method can introduce detectable metabolite-level differences, it does not materially compromise biomarker discovery performance when a single protocol is applied consistently within a study. Thus, any of the three thawing methods can be used in practice, provided consistency is maintained, with the choice guided primarily by logistical considerations.