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BACKGROUND The addition of a calibration curve with every run is both time-consuming and expensive for clinical mass spectrometry assays. We present alternative calibration strategies that eliminate the need for a calibration curve except as required by laboratory regulations. METHODS We measured serum nortriptyline concentrations prospectively in 68 patients on 16 days over a 2-month period using a method employing calibration schemes that relied on the measurement of the response ratio (RR) corrected by the response factor (RF), i.e., a measurement of the RR for an equimolar mixture of the analyte and internal standard. Measurements were taken with contemporaneous RF (cRF) measurements as well as sporadic RF (sRF) measurements. The measurements with these alternative calibration schemes were compared against the clinical results obtained by interpolation on a calibration curve, and those differences were evaluated for analytical and clinical significance. RESULTS The differences between the values measured by cRF and sRF calibration and interpolation on a calibration curve were not significant (P = 0.088 and P = 0.091, respectively). Both the cRF-and sRF-based calibration results demonstrated a low mean bias against the calibration curve interpolations of 3.69% (95% CI, −15.8% to 23.2%) and 3.11% (95% CI, −16.4% to 22.6%), respectively. When these results were classified as subtherapeutic, therapeutic, or supratherapeutic, there was categorical agreement in 95.6% of the cRF calibration results and 94.1% of the sRF results. CONCLUSIONS cRF and sRF calibration in a clinically validated liquid chromatography–tandem mass spectrometry assay yields results that are analytically and clinically commensurate to those produced by interpolation with a calibration curve.

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Problem: Fetal neuroinflammation has been linked to preterm birth-related intraamniotic infection and inflammation; However, the contribution of fetal sex and maternal race/ethnicity is unknown. To determine if fetal sex and maternal race/ethnicity influence neuroinflammation, an organ-on-chip (OOC) model were established under normal or pathologic conditions utilizing amniotic fluid. Method of study:OOC is composed of two-cell culture chambers connected by Type IV collagen-coated microchannels. Human fetal astroglia (SVGp12) and microglia (HMC3) were co-cultured at an 80:20 ratio in the inner chamber. The outer chamber contained amniotic fluid (AF) from male and female fetuses of White Hispanic (WH) and African-American (AA) pregnant women with or without lipopolysaccharide (LPS-100 ng/ml) and incubated for 48 h. Glial migration (brightfield microscopy), activation (Immunocytochemistry), and cytokine production (Luminex assays) were quantified and compared (N = 4 for each category of sex and race/ethnicity). Results:In a pooled analysis, AF+LPS did not induce glial activation or inflammatory changes compared to AF alone. When stratified by sex, male AF+LPS promoted significant glial activation (high CD11b:p < 0.05; low Iba1:p < 0.01) compared to male AF without LPS; however, this was not associated with changes in pro-inflammatory cytokines. When stratified by race/ethnicity, AF+LPS induced glial activation in both groups, but a differential increase in pro-inflammatory cytokines was seen between WH and AA AF (WH-interleukin-1β: p < 0.05; AA-interleukin-8: p < 0.01). Conclusion:This OOC model of fetal neuroinflammation has determined that race/ethnicity differences do exist for perinatal brain injury. The fetal sex of neonates was not a determining factor of susceptibility to intraamniotic inflammation leading to neuroinflammation.