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More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a μLC–MS/MS system

ABSTRACT: Highlights • Sensitive (0.02 ng/mL LLOQ) serum Tg measures by LC–MS/MS at μL/min flow rates.• Tissue-derived Tg can circulate below 0.1 ng/mL in DTC patients with autoantibodies.• Feasibility of Interference-free Tg monitoring below 0.15 ng/mL before recurrence. Although liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays for thyroglobulin (Tg) are resistant to autoantibody (TgAb) interference, recent studies have demonstrated approximately 40% of TgAb-positive individuals with recurrent thyroid cancer have Tg concentrations below the lower limit of quantification (LLOQ) of the LC–MS/MS assays described to date (i.e., <0.5 ng/mL), resulting in false-negative findings during post-thyroidectomy monitoring. To reduce false negative results due to insufficient analytical sensitivity, a new Tg assay was developed on a commercially available LC–MS/MS system operating at microliter/minute flow-rates (i.e., µLC–MS/MS) to maximize the analytical sensitivity and achieve a LLOQ of 0.02 ng/mL. When applied to the measurement of TgAb-negative and TgAb-positive patient serum samples previously measuring below the LLOQ of current immunometric and LC–MS/MS assays (LLOQ, 0.1–0.2 ng/mL), concentrations were measurable by µLC–MS/MS in 66% and 44% of samples, respectively – possibly explaining the persistence of TgAb in those patients. Patients with low Tg concentrations measured by µLC–MS/MS (<0.1 ng/mL) also exhibited elevation in their Tg concentrations upon hormone stimulation, indicating the detected Tg was produced from remnant thyroid tissue and would be suitable as a tissue biomarker. Forty-eight TgAb-positive patient specimens with undetectable Tg by both conventional LC–MS/MS (<0.15 ng/mL) and immunometric (<0.1 ng/mL) assays demonstrated measureable Tg concentrations by the new µLC–MS/MS assay in approximately one-third of the specimens, despite all patients being disease free at the time of collection, suggesting interference-free monitoring of low Tg levels may be feasible prior to the on-set of recurrent disease using a sensitive LC–MS/MS assay.

SUBMITTER: Shuford C

PROVIDER: S-EPMC8601006 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Animal-associated microbes are highly variable, contributing to a diverse set of symbiont-mediated phenotypes. Given that host and symbiont genotypes, and their interactions, can impact symbiont-based phenotypes across environments, there is potential for extensive variation in fitness outcomes. Pea aphids, Acyrthosiphon pisum, host a diverse assemblage of heritable facultative symbionts (HFS) with characterized roles in host defense. Protective phenotypes have been largely studied as single infections, but pea aphids often carry multiple HFS species, and particular combinations may be enriched or depleted compared to expectations based on chance. Here, we examined the consequences of single infection versus coinfection with two common HFS exhibiting variable enrichment, the antiparasitoid Hamiltonella defensa and the antipathogen Regiella insecticola, across three host genotypes and environments. As expected, single infections with either H. defensa or R. insecticola raised defenses against their respective targets. Single infections with protective H. defensa lowered aphid fitness in the absence of enemy challenge, while R. insecticola was comparatively benign. However, as a coinfection, R. insecticola ameliorated H. defensa infection costs. Coinfected aphids continued to receive antiparasitoid protection from H. defensa, but protection was weakened by R. insecticola in two clones. Notably, H. defensa eliminated survival benefits conferred after pathogen exposure by coinfecting R. insecticola Since pathogen sporulation was suppressed by R. insecticola in coinfected aphids, the poor performance likely stemmed from H. defensa-imposed costs rather than weakened defenses. Our results reveal a complex set of coinfection outcomes which may partially explain natural infection patterns and suggest that symbiont-based phenotypes may not be easily predicted based solely on infection status.IMPORTANCE The hyperdiverse arthropods often harbor maternally transmitted bacteria that protect against natural enemies. In many species, low-diversity communities of heritable symbionts are common, providing opportunities for cooperation and conflict among symbionts, which can impact the defensive services rendered. Using the pea aphid, a model for defensive symbiosis, we show that coinfections with two common defensive symbionts, the antipathogen Regiella and the antiparasite Hamiltonella, produce outcomes that are highly variable compared to single infections, which consistently protect against designated enemies. Compared to single infections, coinfections often reduced defensive services during enemy challenge yet improved aphid fitness in the absence of enemies. Thus, infection with multiple symbionts does not necessarily create generalist aphids with "Swiss army knife" defenses against numerous enemies. Instead, particular combinations of symbionts may be favored for a variety of reasons, including their abilities to lessen the costs of other defensive symbionts when enemies are not present.

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More sensitivity is always better: Measuring sub-clinical levels of serum thyroglobulin on a μLC–MS/MS system

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