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.

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.

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.

Point-of-care therapeutic drug monitoring of tumour necrosis factor-α inhibitors using a single step immunoassay.

Therapeutic drug monitoring (TDM) of tumor necrosis factor-α (TNFα)-inhibitors adalimumab and infliximab is important to establish optimal drug dose and maximize treatment efficacy. Currently, TDM is primarily performed with ELISA techniques in clinical laboratories, resulting in a long sample-to-result workflow. Point-of-care (POC) detection of these therapeutic antibodies could significantly decrease turnaround times and allow for user-friendly home-testing. Here, we adapted the recently developed bioluminescent dRAPPID (dimeric Ratiometric Plug-and-Play Immunodiagnostics) sensor platform to allow POC TDM of infliximab and adalimumab. We applied the two best performing dRAPPID sensors, with limit-of-detections of 1 pM and 17 pM, to measure the infliximab and adalimumab levels in 49 and 40 patient serum samples, respectively. The analytical performance of dRAPPID was benchmarked with commercial ELISAs and yielded Pearson's correlation coefficients of 0.93 and 0.94 for infliximab and adalimumab, respectively. Furthermore, a dedicated bioluminescence reader was fabricated and used as a readout device for the TDM dRAPPID sensors. Subsequently, infliximab and adalimumab patient serum samples were measured with the TDM dRAPPID sensors and bioluminescence reader, yielding Pearson's correlation coefficients of 0.97 and 0.86 for infliximab and adalimumab, respectively, and small proportional differences with ELISA (slope was 0.97 ± 0.09 and 0.96 ± 0.20, respectively). The adalimumab and infliximab dRAPPID sensors, in combination with the dedicated bioluminescence reader, allow for ease-of-use TDM with a fast turnaround time and show potential for POC TDM outside of clinical laboratories.

Analysis of Neuron-Specific enolase isozymes in human serum using immunoaffinity purification and liquid chromatography-tandem mass spectrometry quantification.

Neuron-specific enolase (NSE) is a promising small-cell lung cancer (SCLC) biomarker composed of αγ and γγ isozyme dimers. As the conventional immunoassays are prone to interferences and cannot differentiate between the isozymes, we developed a multiplex immunoaffinity (IA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of NSEα and NSEγ in human serum. A calibrator was prepared by performing cold denaturation of recombinantly expressed αα and γγ enolase dimers to induce a new dimer equilibrium that was determined to be approximately 1αγ:1γγ:1αα. Selective sample purification was achieved by performing IA extraction using an antibody specific towards NSEγ. The isolated αγ and γγ dimers were denatured and trypsin digested to allow quantification of the selected signature peptides and their corresponding isotopically labelled peptide internal standard. The obtained linear dynamic ranges were determined to be 1.5-56 ng/mL and 0.64-167 ng/mL for NSEα and NSEγ (R2 = 0.88 and 0.97 respectively). Validation of the assay showed acceptable accuracy and precision for NSEα and NSEγ. The method was successfully applied to patient serum in which both isozymes were detected. Compared to the conventional immunoassay, substantially lower total NSE concentrations were measured in IA LC-MS/MS. With this multiplex IA LC-MS/MS assay, the clinical value of quantifying the individual isozymes can be explored. In addition, together with the calibrator described here, it may be applied to standardize NSE immunoassays across different platforms.

Liquid biopsy-based decision support algorithms for diagnosis and subtyping of lung cancer.

OBJECTIVES: Pathologic subtyping of tissue biopsies is the gold standard for the diagnosis of lung cancer (LC), which could be complicated in cases of e.g. inconclusive tissue biopsies or unreachable tumors. The diagnosis of LC could be supported in a minimally invasive manner using protein tumor markers (TMs) and circulating tumor DNA (ctDNA) measured in liquid biopsies (LBx). This study evaluates the performance of LBx-based decision-support algorithms for the diagnosis of LC and subtyping into small- and non-small-cell lung cancer (SCLC and NSCLC) aiming to directly impact clinical practice. MATERIALS AND METHODS: In this multicenter prospective study (NL9146), eight protein TMs (CA125, CA15.3, CEA, CYFRA 21-1, HE4, NSE, proGRP and SCCA) and ctDNA mutations in EGFR, KRAS and BRAF were analyzed in blood of 1096 patients suspected of LC. The performance of individual and combined TMs to identify LC, NSCLC or SCLC was established by evaluating logistic regression models at pre-specified positive predictive values (PPV) of ≥95% or ≥98%. The most informative protein TMs included in the multi-parametric models were selected by recursive feature elimination. RESULTS: Single TMs could identify LC, NSCLC and SCLC patients with 46%, 25% and 40% sensitivity, respectively, at pre-specified PPVs. Multi-parametric models combining TMs and ctDNA significantly improved sensitivities to 65%, 67% and 50%, respectively. CONCLUSION: In patients suspected of LC, the LBx-based decision-support algorithms allowed identification of about two-thirds of all LC and NSCLC patients and half of SCLC patients. These models therefore show clinical value and may support LC diagnostics, especially in patients for whom pathologic subtyping is impossible or incomplete.

Association of phthalate exposure with thyroid function during pregnancy.

BACKGROUND: The extent of thyroid disruptive effects of phthalates during pregnancy remains unclear. AIM: To investigate the association of maternal urinary phthalates with markers of the thyroid system during early pregnancy. METHODS: Urinary concentrations of phthalate metabolites and serum concentrations of thyroid stimulating hormone (TSH), free and total thyroxine (FT4 and TT4) and free and total triiodothyronine (FT3 and TT3) were measured in pregnant women in early pregnancy in the Swedish Environmental Longitudinal, Mother and child, Asthma and allergy study (2007-ongoing), a population-based prospective cohort. RESULTS: In the 1,996 included women, higher di-ethyl-hexyl phthalate (DEHP) metabolites were associated with a lower FT4 (ß [SE] for the molar sum: -0.13 [0.06], P = 0.03) and a higher TSH/FT4 ratio (0.003 [0.001], P = 0.03). Higher concentrations of di-iso-nonyl phthalate (DINP) metabolites were associated with a lower TT4 (ß [SE] for the molar sum: 0.93 [0.44], P = 0.03) as well as with lower TT4/FT4 and TT4/TT3 ratios. Higher metabolites of both dibutyl and butyl-benzyl phthalate (DBP and BBzP) were associated with lower T4/T3 ratio (free and total) and higher FT4/TT4 and FT3/TT3 ratios. A higher diisononyl cyclohexane dicarboxylate (DINCH) metabolite concentration was associated with a higher TT3. CONCLUSIONS: These results translate results from experimental studies suggesting that exposure to phthalates may interfere with the thyroid system during pregnancy. This is also true for compounds that have been introduced to replace known disruptive phthalates. Further experimental studies should take into account the human evidence to better investigate the potential underlying mechanisms of thyroid disruption by phthalates.

Correction of the NSE concentration in hemolyzed serum samples improves its diagnostic accuracy in small-cell lung cancer.

Neuron-specific enolase (NSE) is a well-known biomarker for the diagnosis, prognosis and treatment monitoring of small-cell lung cancer (SCLC). Nevertheless, its clinical applicability is limited since serum NSE levels are influenced by hemolysis, leading to falsely elevated results. Therefore, this study aimed to develop a hemolysis correction equation and evaluate its role in SCLC diagnostics. Two serum pools were spiked with increasing amounts of hemolysate obtained from multiple individuals. A hemolysis correction equation was obtained by analyzing the relationship between the measured NSE concentration and the degree of hemolysis. The equation was validated using intentionally hemolyzed serum samples, which showed that the correction was accurate for samples with an H-index up to 30 µmol/L. Correction of the measured NSE concentration in patients suspected of lung cancer caused an increase in AUC and a significantly lower cut-off value for SCLC detection when compared to uncorrected results. Therefore, a hemolysis correction equation should be used to correct falsely elevated NSE concentrations. Results of samples with an H-index above 30 µmol/L should not be reported to clinicians. Application of the equation illustrates the importance of hemolysis correction in SCLC diagnostics and questions the correctness of the currently used diagnostic cut-off value.

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.

Reference Ranges and Determinants of Thyroid Function During Early Pregnancy: The SELMA Study.

Context: Establishing reference ranges as well as identifying and quantifying the determinants of thyroid function during pregnancy is important for proper clinical interpretation and optimizing research efforts. However, such data are sparse, specifically for triiodothyronine measurements, and most studies do not take into account thyroid antibodies or human chorionic gonadotropin. Objective: To determine reference ranges and to identify/quantify determinants of TSH, free T4 (FT4), free triiodothyronine (FT3), total T4 (TT4), and total triiodothyronine (TT3). Design, Setting, and Participants: This study included 2314 participants of the Swedish Environmental Longitudinal, Mother and child, Asthma and allergy study, a population-based prospective pregnancy cohort of mother-child pairs. Reference ranges were calculated by 2.5th to 97.5th percentiles after excluding thyroperoxidase antibody (TPOAb)-positive and/or thyroglobulin antibody (TgAb)-positive women. Intervention: None. Main Outcome Measures: TSH, FT4, FT3, TT4, and TT3 in prenatal serum. Results: After exclusion of TPOAb-positive women, reference ranges were as follows: TSH, 0.11 to 3.48 mU/L; FT4, 11.6 to 19.4 pmol/L; FT3, 3.72 to 5.92 pg/mL; TT4, 82.4 to 166.2 pmol/L; and TT3, 1.28 to 2.92 nmol/L. Additional exclusion of TgAb-positive women did not change the reference ranges substantially. Exposure to tobacco smoke, as assessed by questionnaires and serum cotinine, was associated with lower TSH and higher FT3 and TT3. Body mass index (BMI) and gestational age were the main determinants of TSH (only for BMI), FT4, FT3, TT4, and TT3. Conclusions: We show that the exclusion of TgAb-positive women on top of excluding TPOAb-positive women hardly affects clinical reference ranges. We identified various relevant clinical determinants of TSH, FT4, FT3, TT4, and TT3 that could reflect endocrine-disrupting effects and/or effects on thyroid hormone transport or deiodination.

Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins.

BACKGROUND: Given the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. METHODS: By parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and ß-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/ß-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. RESULTS: The α/ß-ratio is a poor marker for thalassemia patients, yet deviant α/ß-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. CONCLUSIONS: Although subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/ß-ratio as screening parameters.

Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer.

The diagnosis of urinary tract infection (UTI) by urine culture is time-consuming and can produce up to 60 to 80% negative results. Fast screening methods that can reduce the necessity for urine cultures will have a large impact on overall turnaround time and laboratory economics. We have evaluated the detection of bacteria and leukocytes by a new urine analyzer, the UF-1000i, to identify negative urine samples that can be excluded from urine culture. In total, 1,577 urine samples were analyzed and compared to urine culture. Urine culture showed growth of ≥10(3) CFU/ml in 939 samples (60%). Receiver operating characteristics (ROC) curves and ROC decision plots were been prepared at three different gold standard definitions of a negative urine culture: no growth, growth of bacteria at <10(4) CFU/ml, and growth of bacteria at <10(5) CFU/ml. Also, the reduction in urine cultures and the percentage of false negatives were calculated. At the most stringent gold standard definition of no growth, a chosen sensitivity of 95% resulted in a cutoff value of 26 bacteria/µl, a specificity of 43% and a reduction in urine cultures of only 20%, of which 14% were false negatives. However, at a gold standard definition of <10(5) CFU/ml and a sensitivity of 95%, the UF-1000i cutoff value was 230 bacteria/µl, the specificity was 80%, and the reduction in urine cultures was 52%, of which 0.3% were false negatives. The applicability of the UF-1000i to screen for negative urine samples strongly depends on population characteristics and the definition of a negative urine culture. In our setting, however, the low workload savings and the high percentage of false-negative results do not warrant the UF-1000i to be used as a screening analyzer.