Immunoaffinity Intact-Mass Spectrometry for the Detection of Endogenous Concentrations of the Acetylated Protein Tumor Biomarker Neuron Specific Enolase.

Intact-mass spectrometry has huge potential for clinical application, as it enables both quantitative and qualitative analysis of intact proteins and possibly unlocks additional pathophysiological information via, e.g., detection of specific post-translational modifications (PTMs). Such valuable and clinically useful selectivity is typically lost during conventional bottom-up mass spectrometry. We demonstrate an innovative immunoprecipitation protein enrichment assay coupled to ultrahigh performance liquid chromatography quadrupole time-of-flight high resolution mass spectrometry (UPLC-QToF-HRMS) for the fast and simple identification of the protein tumor marker Neuron Specific Enolase Gamma (NSEγ) at low endogenous concentrations in human serum. Additionally, using the combination of immunoaffinity purification with intact mass spectrometry, the presence of NSEγ in an acetylated form in human serum was detected. This highlights the unique potential of immunoaffinity intact mass spectrometry in clinical diagnostics.

Machine Learning-Based Prediction of Hemoglobinopathies Using Complete Blood Count Data.

BACKGROUND: Hemoglobinopathies, the most common inherited blood disorder, are frequently underdiagnosed. Early identification of carriers is important for genetic counseling of couples at risk. The aim of this study was to develop and validate a novel machine learning model on a multicenter data set, covering a wide spectrum of hemoglobinopathies based on routine complete blood count (CBC) testing. METHODS: Hemoglobinopathy test results from 10 322 adults were extracted retrospectively from 8 Dutch laboratories. eXtreme Gradient Boosting (XGB) and logistic regression models were developed to differentiate negative from positive hemoglobinopathy cases, using 7 routine CBC parameters. External validation was conducted on a data set from an independent Dutch laboratory, with an additional external validation on a Spanish data set (n = 2629) specifically for differentiating thalassemia from iron deficiency anemia (IDA). RESULTS: The XGB and logistic regression models achieved an area under the receiver operating characteristic (AUROC) of 0.88 and 0.84, respectively, in distinguishing negative from positive hemoglobinopathy cases in the independent external validation set. Subclass analysis showed that the XGB model reached an AUROC of 0.97 for ß-thalassemia, 0.98 for α0-thalassemia, 0.95 for homozygous α+-thalassemia, 0.78 for heterozygous α+-thalassemia, and 0.94 for the structural hemoglobin variants Hemoglobin C, Hemoglobin D, Hemoglobin E. Both models attained AUROCs of 0.95 in differentiating IDA from thalassemia. CONCLUSIONS: Both the XGB and logistic regression model demonstrate high accuracy in predicting a broad range of hemoglobinopathies and are effective in differentiating hemoglobinopathies from IDA. Integration of these models into the laboratory information system facilitates automated hemoglobinopathy detection using routine CBC parameters.

Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS.

OBJECTIVES: Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. METHODS: Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. RESULTS: Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. CONCLUSIONS: A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

High first trimester ferritin levels differ according to parity and are independently related to preterm birth: A prospective cohort study.

INTRODUCTION: A considerable amount of neonatal morbidity and mortality worldwide is caused by preterm birth. To date, the underlying etiology of preterm birth has not been fully clarified. Previous studies demonstrate that inflammation is one of the pathological factors that might cause preterm birth, and that there is a difference between primiparous and multiparous women in immune response to pregnancy. The objective of this prospective cohort study was to investigate the role of two inflammatory markers, ferritin and C-reactive protein (CRP) and preterm birth, in first trimester women, stratified for parity. In addition, a possible association between high ferritin and CRP, and a possible association between high ferritin and CRP and preterm birth were assessed. MATERIAL AND METHODS: A total of 2044 healthy, low-risk pregnant women from primary obstetric care in the Netherlands participated in this study. Their ferritin and CRP levels were evaluated at 12 weeks' gestation. Levels above the parity specific 95th percentile were defined as high. The main outcome of this study was to assess the presence of a possible association between parity specific high ferritin and CRP, and preterm birth. The secondary outcomes were the ferritin and CRP levels of women, stratified for parity, and the possible association between high ferritin and CRP levels. Logistic regression analysis was performed with preterm birth as a dependent variable and parity specific high ferritin and CRP as an independent variable, adjusting for age and history of preterm birth. RESULTS: Ferritin levels decreased with increasing parity. Ferritin and CRP levels at 12 weeks' gestation were significantly higher in women with preterm birth. In primiparous women, high ferritin levels (OR: 2.5, CI: 1.14-5.38) and high CRP levels (OR: 5.0, CI: 2.61-9.94) were independently associated with preterm birth. In multiparous women, high ferritin levels (OR: 6.0, CI: 2.28-16.67) were independently associated with preterm birth while high CRP levels were not. CONCLUSIONS: First trimester parity specific ferritin and CRP levels could play a part in predictive models for preterm birth, and further research for their additive role in preterm birth is needed.

Hormonal and psychological factors in nausea and vomiting during pregnancy.

BACKGROUND: The aetiology of nausea and vomiting during pregnancy (NVP) is multifactorial, but the relative contribution of biological and psychological determinants is insufficiently understood. We examined the association of human chorionic gonadotropin (hCG), thyroid hormones (thyroid-stimulating hormone and thyroxin) and psychological factors with NVP. METHODS: Blood chemistry and psychological measures were obtained in 1682 pregnant women participating in the Holistic Approach to Pregnancy and the first Postpartum Year (HAPPY) study between 12 and 14 weeks of gestation. The presence of NVP was measured using the Pregnancy-Unique Quantification of Emesis scale. Depressive symptoms were assessed using the Edinburgh Depression Scale. Multivariable logistic regression analyses were used to investigate the independent role of hCG, thyroid hormones and depression as related to NVP, adjusting for age, body mass index, education, parity, smoking status, unplanned pregnancy and history of depression. RESULTS: Elevated levels of NVP were observed in 318 (18.9%) participants. High hCG levels [odds ratio (OR) = 1.47, 95% confidence interval (CI) = 1.11-1.95], elevated depressive symptoms in the first trimester (OR = 1.67, 95% CI = 1.15-2.43) and a history of depression (OR = 1.53, 95% CI = 1.11-2.11) were independently related to high NVP. Multiparity (OR = 1.47, 95% CI = 1.12-1.92) and younger age (OR = 0.91, 95% CI = 0.87-0.94) were also associated with high NVP, whereas (sub)clinical hyperthyroidism was not related to high NVP. CONCLUSIONS: The current study is the first to demonstrate that a combination of hCG hormone and psychological factors are independently related to nausea and vomiting during early pregnancy.

Maternal cognitive function during pregnancy in relation to hypo- and hyperthyroxinemia.

OBJECTIVE: To assess a possible relationship between maternal cognitive dysfunction during pregnancy and hypothyroxinemia, adjusted for major confounders. BACKGROUND: Thyroid dysfunction in general is associated with cognitive dysfunction. Cognitive dysfunction is common during pregnancy. DESIGN: Prospective follow-up study from 12 to 32 weeks of pregnancy. PARTICIPANTS: 2082 healthy pregnant women. MEASUREMENTS: Cognitive function, depression and sleeping problems were assessed by self-report questionnaires at 12, 22 and 32 weeks of gestation, higher scores reflecting more symptoms. FT4, TSH and TPO-Ab were assessed at 12 weeks of gestation. DEFINITIONS: healthy (euthyroxinemia) control group: FT4 within 10-90th percentiles, without elevated TPO-Ab titres and TSH within first trimester-specific reference range (0.23-4.0 mU/L). Hypothyroxinemia: FT4 <2.5th percentile with TSH within first trimester-specific reference range. Poor cognitive function: a score >1 SD > mean on the cognitive function scale. RESULTS: A total of 54 women showed hypothyroxinemia and 1476 women had euthyroxinemia. At 12 weeks, multiple logistic regression showed that poor cognitive function was independently related to hypothyroxinemia: OR: 2.9 (95% CI: 1.6-5.4), adjusted for depression (OR: 3.1; 95% CI: 2.7-4.6) and sleeping problems (OR: 2.8, 95% CI: 1.9-3.9). TPO-Ab + women with hypothyroxinemia had the highest levels of cognitive dysfunction. Other cut-offs of hypothyroxinemia (<5th or <10th percentile with normal TSH) showed similar results. GLM-ANOVA showed that throughout pregnancy women with hypothyroxinemia at 12 weeks had significantly higher cognitive dysfunction scores compared with the healthy controls: F = 12.1, P = .001. CONCLUSIONS: Women with hypothyroxinemia during early gestation are at risk for poor cognitive function throughout gestation, adjusted for depression and sleeping problems.

Urine flow cytometry is an adequate screening tool for urinary tract infections in children.

Diagnosing a urinary tract infection in children is often difficult due to non-specific symptoms and requires invasive and time-consuming procedures. Flow cytometry is a new and rapid method of analyzing urine to confirm or exclude UTIs. We have investigated the sensitivity and specificity of urine flow cytometry (Sysmex UF1000i) compared to conventional diagnostic techniques in a prospective study from January 1, 2014 until January 1, 2015. All children under 13 years of age with a suspicion of urinary tract infection were screened using both urine flow cytometry and urine culture. A urinary tract infection was defined as the combination of leukocyturia (≥ 25 leukocytes per µl) and a positive urine culture in the presence of clinical symptoms. A total number of 412 urine samples were collected, of which 63 cases (15.3%) were positive for a urinary tract infection. Receiver operating characteristic analysis showed an area under the curve of 0.97 (95% confidence interval h0.93-1.00) for the bacterial count. When using a cut-off value of 250 bacteria/µl in the presence of leukocyturia, the sensitivity for urinary tract infection is 0.97 with a negative predictive value of 97%, and the specificity is 0.91 with a positive predictive value of 90%.Conclusion: Flow cytometry-based bacterial and leukocyte count analysis is a time-efficient method of diagnosing or ruling out urinary tract infection in children, with a higher sensitivity and specificity than dipstick and microscopic analysis. What is known • Screening for urinary tract infections in children is difficult due to invasive and time-consuming procedures. • There is both over- and under-treatment of urinary tract infections due to the delays in accurate diagnosing. What is new • Flow cytometry is a rapid and accurate method to provide useful information in the diagnosis of urinary tract infection in children. When negative, flow cytometry can exclude urinary tract infection in children with a high degree of confidence. When flow cytometry is positive, the possibility of a urinary tract infection in children is increased.

Optimizing charge state distribution is a prerequisite for accurate protein biomarker quantification with LC-MS/MS, as illustrated by hepcidin measurement.

BACKGROUND: Targeted quantification of protein biomarkers with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has great potential, but is still in its infancy. Therefore, we elucidated the influence of charge state distribution and matrix effects on accurate quantification, illustrated by the peptide hormone hepcidin. METHODS: An LC-MS/MS assay for hepcidin, developed based on existing literature, was improved by using 5 mM ammonium formate buffer as mobile phase A and as an elution solution for solid phase extraction (SPE) to optimize the charge state distribution. After extensive analytical validation, focusing on interference and matrix effects, the clinical consequence of this method adjustment was studied by performing receiving operating characteristic (ROC)-curve analysis in patients with iron deficiency anemia (IDA, n=44), anemia of chronic disease (ACD, n=42) and non-anemic patients (n=93). RESULTS: By using a buffered solution during sample preparation and chromatography, the most abundant charge state was shifted from 4+ to 3+ and the charge state distribution was strongly stabilized. The matrix effects which occurred in the 4+ state were therefore avoided, eliminating bias in the low concentration range of hepcidin. Consequently, sensitivity, specificity and positive predictive value (PPV) for detection of IDA patients with the optimized assay (96%, 97%, 91%, respectively) were much better than for the original assay (73%, 70%, 44%, respectively). CONCLUSIONS: Fundamental improvements in LC-MS/MS assays greatly impact the accuracy of protein quantification. This is urgently required for improved diagnostic accuracy and clinical value, as illustrated by the validation of our hepcidin assay.

Therapeutic drug monitoring of infliximab: performance evaluation of three commercial ELISA kits.

BACKGROUND: Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. METHODS: The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 µg/mL and determining assay concordance. RESULTS: Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. CONCLUSIONS: Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardization.

The HAPPY study (Holistic Approach to Pregnancy and the first Postpartum Year): design of a large prospective cohort study.

BACKGROUND: The HAPPY study is a large prospective longitudinal cohort study in which pregnant women (N ≈ 2,500) are followed during the entire pregnancy and the whole first year postpartum. The study collects a substantial amount of psychological and physiological data investigating all kinds of determinants that might interfere with general well-being during pregnancy and postpartum, with special attention to the effect of maternal mood, pregnancy-related somatic symptoms (including nausea and vomiting (NVP) and carpal tunnel syndrome (CTS) symptoms), thyroid function, and human chorionic gonadotropin (HCG) on pregnancy outcome of mother and foetus. METHODS/DESIGN: During pregnancy, participants receive questionnaires at 12, 22 and 32 weeks of gestation. Apart from a previous obstetric history, demographic features, distress symptoms, and pregnancy-related somatic symptoms are assessed. Furthermore, obstetrical data of the obstetric record form and ultrasound data are collected during pregnancy. At 12 and 30 weeks, thyroid function is assessed by blood analysis of thyroid stimulating hormone (TSH), free thyroxine (FT4) and thyroid peroxidase antibodies (TPO-Ab), as well as HCG. Also, depression is assessed with special focus on the two key symptoms: depressed mood and anhedonia. After childbirth, cord blood, neonatal heel screening results and all obstetrical data with regard to start of labour, mode of delivery and complications are collected. Moreover, mothers receive questionnaires at one week, six weeks, four, eight, and twelve months postpartum, to investigate recovery after pregnancy and delivery, including postpartum mood changes, emotional distress, feeding and development of the newborn. DISCUSSION: The key strength of this large prospective cohort study is the holistic (multifactorial) approach on perinatal well-being combined with a longitudinal design with measurements during all trimesters of pregnancy and the whole first year postpartum, taking into account two physiological possible markers of complaints and symptoms throughout gestation: thyroid function and HCG. The HAPPY study is among the first to investigate within one design physiological and psychological aspects of NVP and CTS symptoms during pregnancy. Finally, the concept of anhedonia and depressed mood as two distinct aspects of depression and its possible relation on obstetric outcome, breastfeeding, and postpartum well-being will be studied.

Hypothyroxinaemia during gestation is associated with low ferritin and increased levels of inflammatory markers.

Objective: Pregnancy is a state of physiological inflammation facilitating implantation. Early isolated hypothyroxinaemia (IH) and increased inflammation (including obesity) have been associated with severe obstetric complications. The current study evaluated the association between IH, low ferritin and inflammation parameters (interleukin 6 (IL-6), C-reactive protein (CRP), human chorionic gonadotrophin (hCG) and obesity. Moreover, the course of these parameters throughout pregnancy was evaluated in relation to IH. Methods: In the cross-sectional study (A) at 12 weeks, 2759 women participated and 2433 participated in the longitudinal study (B) with assessments at 12, 20 and 28 weeks gestation. At the first trimester, 122 (4.4%) IH women (free thyroxine (FT4) <5th percentile, normal TSH levels) were compared with 2114 (76.6%) reference women (FT4 between tenth and 90th percentiles, normal thyrotrophin (TSH) levels), in study B these figures were 99 (4.1%) and 1847 (75.9%), respectively. Results: Cross-sectionally, compared to reference women, IH was independently associated with low ferritin (<5th percentile, OR: 2.6, 95% CI: 1.4-4.9), high CRP (>95th percentile: OR: 1.9, 95% CI: 1.04-3.7), low hCG ( 30, OR: 1.7, 95% CI: 1.12.9) and higher age (OR: 1.1, 95% CI: 1.04-1.15). Longitudinally, compared to reference women, women with IH at 12 weeks gestation showed persistently and significantly lower ferritin and hCG levels, and persistently higher CRP and IL-6 levels throughout gestation. Conclusion: Gestational IH could be viewed as a condition of increased inflammation, as reported in non-thyroidal illness syndrome. Less favourable inflammation parameters and low iron status during early gestation in IH women seem to persist throughout gestation.

Partial protein binding of uracil and thymine affects accurate dihydropyrimidine dehydrogenase (DPD) phenotyping.

Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60-65 %) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.

Strengths and challenges in current lung cancer care: Timeliness and diagnostic procedures in six Dutch hospitals.

OBJECTIVES: Timely diagnosis of lung cancer (LC) is crucial to achieve optimal patient care and outcome. Moreover, the number of procedures required to obtain a definitive diagnosis can have a large influence on the life expectancy of a patient. Here, adherence with existing Dutch guidelines for timeliness and type and number of invasive and imaging procedures was assessed. MATERIALS AND METHODS: 1096 patients with suspected LC were enrolled in this multicenter prospective study (NL9146). The overall survival, time from referral to the first appointment with the pulmonologist, time to diagnosis and treatment, and the number of imaging and invasive procedures were evaluated. Patients were divided into different diagnostic groupsearly- and advanced stage non-small-cell lung cancer (NSCLC), small-cell lung cancer (SCLC), large cell neuroendocrine carcinoma of the lung (LCNEC), patients without LC and patients without a definitive diagnosis. RESULTS: The majority of patients (66 %) received a definitive diagnosis within 5 weeks, although the time to diagnosis of early-stage LC patients and patients without LC was significantly longer comparted to advanced stage LC. An increase in invasive procedures was seen for early-stage LC compared to advanced stage LC and for 13 % of the advanced stage non-squamous NSCLC patients up to three additional invasive procedures were performed solely to obtain sufficient material for NGS. For patients without a definitive diagnosis, 50 % did undergo at least one invasive procedure, while 11 % did not wish to undergo any invasive procedures. CONCLUSION: These insights could aid in improved LC diagnostics and efficient implementation of new techniques like liquid biopsy and artificial intelligence. This may lead to more timely LC care, a decreased number of invasive procedures, less variability between the diagnostic trajectory of different patients and aid in obtaining a definitive diagnosis for all patients.

Association between per- and polyfluoroalkyl substances exposure and thyroid function biomarkers among females attending a fertility clinic.

Per- and polyfluoroalkyl substances (PFAS) exposure was associated with changes in thyroid function in pregnant mothers and the general population. Limited such evidence exists in other susceptible populations such as females with fertility problems. This cross-sectional study included 287 females seeking medically assisted reproduction at a fertility clinic in Massachusetts, United States, between 2005 and 2019. Six long-alkyl chain PFAS, thyroid hormones, and autoimmune antibodies were quantified in baseline serum samples. We used generalized linear models and quantile g-computation to evaluate associations of individual PFAS and their total mixture with thyroid biomarkers. Most females were White individuals (82.7%), had graduate degrees (57.8%), and nearly half had unexplained subfertility (45.9%). Serum concentrations of all examined PFAS and their mixture were significantly associated with 2.6%-5.6% lower total triiodothyronine (TT3) concentrations. Serum concentrations of perfluorononanoate (PFNA), perfluorodecanoate (PFDA), and perfluoroundecanoate (PFUnDA), and of the total mixture were associated with higher ratios of free thyroxine (FT4) to free triiodothyronine (FT3). No associations were found for PFAS and TSH or autoimmune antibodies. Our findings support the thyroid-disrupting effect of long alkyl-chain PFAS among a vulnerable population of subfertile females.

TSH and FT4 Reference Interval Recommendations and Prevalence of Gestational Thyroid Dysfunction: Quantification of Current Diagnostic Approaches.

CONTEXT: Guidelines recommend use of population- and trimester-specific thyroid-stimulating hormone (TSH) and free thyroxine (FT4) reference intervals (RIs) in pregnancy. Since these are often unavailable, clinicians frequently rely on alternative diagnostic strategies. We sought to quantify the diagnostic consequences of current recommendations. METHODS: We included cohorts participating in the Consortium on Thyroid and Pregnancy. Different approaches were used to define RIs: a TSH fixed upper limit of 4.0 mU/L (fixed limit approach), a fixed subtraction from the upper limit for TSH of 0.5 mU/L (subtraction approach) and using nonpregnancy RIs. Outcome measures were sensitivity and false discovery rate (FDR) of women for whom levothyroxine treatment was indicated and those for whom treatment would be considered according to international guidelines. RESULTS: The study population comprised 52 496 participants from 18 cohorts. Compared with the use of trimester-specific RIs, alternative approaches had a low sensitivity (0.63-0.82) and high FDR (0.11-0.35) to detect women with a treatment indication or consideration. Sensitivity and FDR to detect a treatment indication in the first trimester were similar between the fixed limit, subtraction, and nonpregnancy approach (0.77-0.11 vs 0.74-0.16 vs 0.60-0.11). The diagnostic performance to detect overt hypothyroidism, isolated hypothyroxinemia, and (sub)clinical hyperthyroidism mainly varied between FT4 RI approaches, while the diagnostic performance to detect subclinical hypothyroidism varied between the applied TSH RI approaches. CONCLUSION: Alternative approaches to define RIs for TSH and FT4 in pregnancy result in considerable overdiagnosis and underdiagnosis compared with population- and trimester-specific RIs. Additional strategies need to be explored to optimize identification of thyroid dysfunction during pregnancy.

Defining gestational thyroid dysfunction through modified non-pregnancy reference intervals: an individual participant meta-analysis.

BACKGROUND: Establishing local trimester-specific reference intervals for gestational TSH and FT4 is often not feasible, necessitating alternative strategies. We aimed to systematically quantify the diagnostic performance of standardized modifications of center-specific non-pregnancy reference intervals as compared to trimester-specific reference intervals. METHODS: We included prospective cohorts participating in the Consortium on Thyroid and Pregnancy. After relevant exclusions, reference intervals were calculated per cohort in thyroperoxidase antibody-negative women. Modifications to the non-pregnancy reference intervals included an absolute modification (per 0.1 mU/L TSH or 1 pmol/L FT4), relative modification (in steps of 5%) and fixed limits (upper TSH limit between 3.0 to 4.5 mU/L and lower FT4 limit 5-15 pmol/L). We compared (sub)clinical hypothyroidism prevalence, sensitivity and positive predictive value (PPV) of aforementioned methodologies with population-based trimester-specific reference intervals. RESULTS: The final study population comprised 52,496 participants in 18 cohorts. Optimal modifications of standard reference intervals to diagnose gestational overt hypothyroidism were -5% for the upper limit of TSH and +5% for the lower limit of FT4 (sensitivity 0.70, confidence interval [CI] 0.47-0.86; PPV 0.64, CI 0.54-0.74). For subclinical hypothyroidism, these were -20% for the upper limit of TSH and -15% for the lower limit of FT4 (sensitivity 0.91, CI 0.67-0.98; PPV 0.71, CI 0.58-0.80). Absolute and fixed modifications yielded similar results. Confidence intervals were wide, limiting generalizability. CONCLUSION: We could not identify modifications of non-pregnancy TSH and FT4 reference intervals that would enable centers to adequately approximate trimester-specific reference intervals. Future efforts should be turned towards studying the meaningfulness of trimester-specific reference intervals and risk-based decision limits.

Risk Factors for Thyroid Dysfunction in Pregnancy: An Individual Participant Data Meta-Analysis.

Background: International guidelines recommend targeted screening to identify gestational thyroid dysfunction. However, currently used risk factors have questionable discriminative ability. We quantified the risk for thyroid function test abnormalities for a subset of risk factors currently used in international guidelines. Methods: We included prospective cohort studies with data on gestational maternal thyroid function and potential risk factors (maternal age, body mass index [BMI], parity, smoking status, pregnancy through in vitro fertilization, twin pregnancy, gestational age, maternal education, and thyroid peroxidase antibody [TPOAb] or thyroglobulin antibody [TgAb] positivity). Exclusion criteria were pre-existing thyroid disease and use of thyroid interfering medication. We analyzed individual participant data using mixed-effects regression models. Primary outcomes were overt and subclinical hypothyroidism and a treatment indication (defined as overt hypothyroidism, subclinical hypothyroidism with thyrotropin >10 mU/L, or subclinical hypothyroidism with TPOAb positivity). Results: The study population comprised 65,559 participants in 25 cohorts. The screening rate in cohorts using risk factors currently recommended (age >30 years, parity ≥2, BMI ≥40) was 58%, with a detection rate for overt and subclinical hypothyroidism of 59%. The absolute risk for overt or subclinical hypothyroidism varied <2% over the full range of age and BMI and for any parity. Receiver operating characteristic curves, fitted using maternal age, BMI, smoking status, parity, and gestational age at blood sampling as explanatory variables, yielded areas under the curve ranging from 0.58 to 0.63 for the primary outcomes. TPOAbs/TgAbs positivity was associated with overt hypothyroidism (approximate risk for antibody negativity 0.1%, isolated TgAb positivity 2.4%, isolated TPOAb positivity 3.8%, combined antibody positivity 7.0%; p < 0.001), subclinical hypothyroidism (risk for antibody negativity 2.2%, isolated TgAb positivity 8.1%, isolated TPOAb positivity 14.2%, combined antibody positivity 20.0%; p < 0.001) and a treatment indication (risk for antibody negativity 0.2%, isolated TgAb positivity 2.2%, isolated TPOAb positivity 3.0%, and combined antibody positivity 5.1%; p < 0.001). Twin pregnancy was associated with a higher risk of overt hyperthyroidism (5.6% vs. 0.7%; p < 0.001). Conclusions: The risk factors assessed in this study had poor predictive ability for detecting thyroid function test abnormalities, questioning their clinical usability for targeted screening. As expected, TPOAb positivity (used as a benchmark) was a relevant risk factor for (subclinical) hypothyroidism. These results provide insights into different risk factors for gestational thyroid dysfunction.

The first trimester plasma copper-zinc ratio is independently related to pregnancy-specific psychological distress symptoms throughout pregnancy.

OBJECTIVES: High plasma copper (Cu) and low zinc (Zn) levels have been associated with depression. However, most studies used low sample sizes and a cross-sectional design, and perinatal data are scarce. We investigated the possible association between pregnancy-specific psychological distress and the plasma CuZn ratio using a prospective design. METHODS: Pregnancy-specific distress symptoms were assessed at each trimester by means of the Tilburg Pregnancy Distress Scale, negative affect subscale, in 2036 pregnant women. Cu and Zn were assessed at 12 wk of gestation in plasma samples by inductively coupled plasma mass spectrometry. Growth mixture modeling determined trajectories of women's pregnancy-specific negative affect (P-NA) symptoms, which were entered in a multiple logistic regression analysis as dependent variable and the CuZn ratio as independent variable. RESULTS: Two P-NA symptom classes were found: 1) persistently low (n = 1820) and 2) persistently high (n = 216). A higher CuZn ratio was independently associated with persistently high P-NA symptom scores (odds ratio = 1.52; 95% confidence interval, 1.13-2.04) after adjustment for confounders. A sensitivity analysis was performed excluding all women with high P-NA scores at 12 wk of gestation (>1 SD above the mean P-NA score). In the 1719 remaining women, a higher CuZn ratio significantly predicted the development of increasing P-NA symptom scores after adjustment for confounders (odds ratio = 1.40; 95% confidence interval, 1.04-1.95). CONCLUSIONS: A higher CuZn plasma ratio is an independent determinant of developing pregnancy-specific distress symptoms throughout pregnancy, suggesting that micronutrients could be used as novel biomarkers for psychological distress research of perinatal mood disorders.

Association of endocrine disrupting chemicals exposure with human chorionic gonadotropin concentrations in pregnancy.

BACKGROUND: Human chorionic gonadotropin (hCG) is produced by the placenta and plays an essential role in the maintenance of pregnancy. Endocrine disrupting chemicals (EDCs) have the potential to interfere with functions related to the production and secretion of hCG; however associations between exposure to EDCs and hCG concentrations in humans remain to be elucidated. OBJECTIVES: To investigate the association of urinary, serum and plasma concentrations of EDCs during pregnancy with serum hCG concentrations. METHODS: We utilized data form the Swedish Environmental Longitudinal, Mother and child, Asthma and allergy (SELMA) study. We investigated the association of 26 EDCs measured in early pregnancy urine or blood with serum hCG concentrations using multi-variable adjusted linear regression models per EDC and Weighted Quantile Sum (WQS) regression with repeated holdout validation for the EDCs mixture. RESULTS: In 2,039 included women, higher exposure to bisphenol A was associated with lower hCG (beta [95% CI]: -0.06 [-0.11 to -0.002]) while higher triclosan exposure was associated with a higher hCG (0.02 [0.003 to 0.04]). Higher exposure to several phthalates, including mono-ethyl and mono-butyl phthalates (MEP and MBP) as well as metabolites of di-2-ethylhexyl phthalate (DEHP) was associated with a lower hCG (beta [95% CI] for sum of DEHP metabolites: -0.13 [-0.19 to -0.07]). Likewise, higher exposure to several polychlorinated biphenyls (PCBs) was associated with a lower hCG. In the WQS regression, each quartile increase in the EDCs mixture was associated with -0.27 lower hCG (95% CI: -0.34 to -0.19). DISCUSSION: Higher exposure to several EDCs during pregnancy was associated with a lower hCG; and despite the small effect sizes, still indicating that the exposure may negatively affect production or secretion of hCG by the placenta. Our results provide the impetus for future experimental studies to investigate the placenta as a target organ for adverse effects of EDCs.

Up-front mutation detection in circulating tumor DNA by droplet digital PCR has added diagnostic value in lung cancer.

Identification of actionable mutations in advanced stage non-squamous non-small-cell lung cancer (NSCLC) patients is recommended by guidelines as it enables treatment with targeted therapies. In current practice, mutations are identified by next-generation sequencing of tumor DNA (tDNA-NGS), which requires tissue biopsies of sufficient quality. Alternatively, circulating tumor DNA (ctDNA) could be used for mutation analysis. This prospective, multicenter study establishes the diagnostic value of ctDNA analysis by droplet digital PCR (ctDNA-ddPCR) in patients with primary lung cancer. CtDNA from 458 primary lung cancer patients was analyzed using a panel of multiplex ddPCRs for EGFR (Ex19Del, G719S, L858R, L861Q and S768I), KRAS G12/G13 and BRAF V600 mutations. For 142 of 175 advanced stage non-squamous NSCLC patients tDNA-NGS results were available to compare to ctDNA-ddPCR. tDNA-NGS identified 98 mutations, of which ctDNA-ddPCR found 53 mutations (54%), including 32 of 45 (71%) targetable driver mutations. In 2 of these 142 patients, a mutation was found by ctDNA-ddPCR only. In 33 advanced stage patients lacking tDNA-NGS results, ctDNA-ddPCR detected 15 additional mutations, of which 7 targetable. Overall, ctDNA-ddPCR detected 70 mutations and tDNA-NGS 98 mutations in 175 advanced NSCLC patients. Using an up-front ctDNA-ddPCR strategy, followed by tDNA-NGS only if ctDNA-ddPCR analysis is negative, increases the number of mutations found from 98 to 115 (17%). At the same time, up-front ctDNA-ddPCR reduces tDNA-NGS analyses by 40%, decreasing the need to perform (additional) biopsies.